U.S. patent number 5,876,979 [Application Number 08/485,778] was granted by the patent office on 1999-03-02 for rna component of mouse, rat, chinese hamster and bovine telomerase.
This patent grant is currently assigned to Cold Spring Harbor Laboratory. Invention is credited to William H. Andrews, Ariel Athena Avilion, Junli Feng, Walter Funk, Carol Greider, Maria Antonia Blasco Marhuenda, Bryant Villeponteau.
United States Patent |
5,876,979 |
Andrews , et al. |
March 2, 1999 |
**Please see images for:
( Certificate of Correction ) ** |
RNA component of mouse, rat, Chinese hamster and bovine
telomerase
Abstract
Nucleic acids comprising the RNA component of a mouse, rat,
Chinese hamster and bovine telomerase are disclosed, as are
recombinant expression plasmids comprising said nucleic acids and
host cells transformed with said recombinant expression
plasmids.
Inventors: |
Andrews; William H. (Richmond,
CA), Avilion; Ariel Athena (London, GB), Feng;
Junli (San Carlos, CA), Funk; Walter (Union City,
CA), Greider; Carol (Huntington, NY), Marhuenda; Maria
Antonia Blasco (Mill Neck, NY), Villeponteau; Bryant
(San Carlos, CA) |
Assignee: |
Cold Spring Harbor Laboratory
(Cold Spring Harbor, NY)
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Family
ID: |
27501039 |
Appl.
No.: |
08/485,778 |
Filed: |
June 7, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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387524 |
Feb 13, 1995 |
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330123 |
Oct 27, 1994 |
5583016 |
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272102 |
Jul 7, 1994 |
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Current U.S.
Class: |
435/91.3;
435/325; 435/320.1; 536/24.3; 536/24.5; 536/23.2; 536/23.1 |
Current CPC
Class: |
C12Q
1/68 (20130101); A61P 35/00 (20180101); C12Y
207/07049 (20130101); C12N 15/113 (20130101); C12N
9/1241 (20130101); C12N 15/1137 (20130101); C12Q
1/6886 (20130101); C12Q 1/6806 (20130101); A61P
31/12 (20180101); C12N 15/11 (20130101); C12Q
1/6841 (20130101); C12Q 2600/158 (20130101); C12N
2310/315 (20130101); A01K 2217/05 (20130101); C12N
2310/15 (20130101); C12N 2310/321 (20130101); C12N
2310/111 (20130101); C12N 2310/121 (20130101); C12N
2310/13 (20130101); C12N 2310/321 (20130101); C12N
2310/3521 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12N 15/11 (20060101); C12N
9/12 (20060101); C07H 021/04 (); C12N 005/16 ();
C12N 015/52 (); C12N 015/85 () |
Field of
Search: |
;536/23.1,23.2,24.3,24.5
;435/320.1,325,172.3,183,91.3 |
References Cited
[Referenced By]
U.S. Patent Documents
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5583016 |
December 1996 |
Villeponteau et al. |
5585479 |
December 1996 |
Hoke et al. |
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Foreign Patent Documents
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0666313A2 |
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Jan 1995 |
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EP |
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93/23572 |
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Nov 1993 |
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WO |
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95/13383 |
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May 1995 |
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WO |
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95/13382 |
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May 1995 |
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WO |
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95/13381 |
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May 1995 |
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WO |
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Other References
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Morin, Gregg, B., "The Human Telomere Terminal Transferase Enzyme
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59:521-529 (1989). .
Romero and Blackburn, "A Conserved Secondary Structure for
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Singer and Gottschling, "TLC1: Template RNA Component of
Saccharomyces cerevisiae," Science, 266:404-409 (1994). .
Counter, Christopher M., et al., "Telomere Shortening Associated
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Collins and Greider, "Tetrahymena Telomerase Catalyzes Nucleolytic
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Mutant Tetrahymena Telomerase," Genes & Development, 8:563-575
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Collins, Kathleen, et al., "Purification of Tetrahymena Telomerase
and Cloning of Genes Encoding the Two Protein Components of the
Enzyme," Cell, 81:677-686 (1995). .
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Science, 269:1236-1241 (1995). .
Blackburn, E.H., "Telomerases," Annun. Rev. Biochem., 61:113-129
(1992). .
Morin, G.B., "Recognition of a Chromosome Truncation Site
Associated with .alpha.-thalassaemia by Human Telomerase," Nature,
353:454-456 (1991). .
Harley, C.B., "Telomere Loss: Mitotic Clock or Genetic Time Bomb?,"
Mutation Research, 256:271-282 (1991). .
Yu, Guo-Liang, et al., "In Vivo Alteration of Telomere Sequences
and Senescence Caused by Mutated Tetrahymena Telomerase and Cloning
of Genes Encoding the Two Protein Components of the Enzyme," Cell,
81:677-686 (1995). .
Blasco, M.A., et al., "Functional Characterization and
Developmental Regulation of Mouse Telomerase RNA," Science,
269:1267-1270 (1995). .
Preker P, et al. "Mapping and characterization of the promoter
elements of the regulatory nif genes rpoN, nifA1 and nifA2 in
Rhodobacter capsulatus." Mol. Microbiol. 6: 1035-1047, 1992. .
Selbie LA, et al. "The major dopamine D2 receptor: molecular
anaylsis of the human D2A subtype." DNA 8:683-689, 1989. .
Sambrook J, et al. "Molecular Cloning." Cold Spring Harbor
Laboratory Press, NY, pp. 18.82-18.84, 1989. .
Harrington, L.A., "Characterization and Purification of Tetrahymena
Telomerase," A Ph.D. Thesis presented at the State University of
New York at Stony Brook, pp. 112-194 and 201-205 (Dec. 1993). .
Strahl, C. et al., "The Effects of Nucleoside Analogs on Telomerase
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Blackburn, E.H., "Structure and Function of Telomeres," Nature
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Primary Examiner: Robinson; Douglas W.
Assistant Examiner: Nelson; Amy J.
Attorney, Agent or Firm: Hamilton, Brook, Smith &
Reynolds, P.C.
Government Interests
GOVERNMENT SUPPORT
The invention described herein was made in whole or in part with
government support under Grants Number NIH 5R01 GM43080-05 and
Number NIH 5R01AG09383-04 awarded by the National Institutes of
Health. The United States Government has certain right in the
invention.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation-in-part of U.S. patent
application Ser. No. 08/387,524, filed Feb. 13, 1995, now
abandoned, which is a continuation-in-part of U.S. patent
application Ser. No. 08/330,123 filed Oct. 27, 1994, now U.S. Pat.
No. 5,583,016, which is a continuation-in-part of U.S. patent
application Ser. No. 08/272,102, filed Jul. 7, 1994 now abandoned.
The teachings of all of these applications are expressly
incorporated herein by reference.
Claims
We claim:
1. An isolated RNA comprising the RNA component of mouse
telomerase.
2. The isolated RNA of claim 1 having a sequence identical to SEQ
ID NO:4.
3. An isolated RNA comprising the RNA component of rat
telomerase.
4. The isolated RNA of claim 3 having a sequence encoded by SEQ ID
NO:5.
5. An isolated RNA comprising the RNA component of Chinese hamster
telomerase.
6. The isolated RNA of claim 5 having a sequence encoded by SEQ ID
NO:43.
7. An isolated RNA comprising the RNA component of bovine
telomerase.
8. The isolated RNA of claim 7 having a sequence encoded by SEQ ID
NO:44.
9. An isolated DNA that encodes the isolated RNA of any one of
claims 1, 3, 5 and 7.
10. A recombinant expression plasmid comprising the DNA of claim 9
and further comprising a promoter positioned to drive transcription
of an RNA encoded by said DNA.
11. A host cell transformed with the recombinant expression plasmid
of claim 10, wherein said plasmid functions to produce the RNA in
said host cell.
12. An oligonucleotide comprising or encoding 50 or more
consecutive nucleotides of the isolated RNA claim 2, or an
oligonucleotide complementary to 50 or more consecutive nucleotides
of the isolated RNA of claim 2.
13. The oligonucleotide of claim 12 that is an
oligodeoxyribonucleotide.
14. The oligonucleotide of claim 12 that is an
oligoribonucleotide.
15. The oligonucleotide of claim 12 that, when bound to an RNA
component of mouse telomerase, inhibits or blocks the activity of
the telomerase.
16. A recombinant expression plasmid comprising the oligonucleotide
of claim 13 and further comprising a promoter positioned to drive
transcription of an RNA encoded by said oligonucleotide.
17. The recombinant expression plasmid of claim 16, wherein said
plasmid functions to produce the RNA in eukaryotic host cells.
18. The recombinant expression plasmid of claim 16, wherein said
plasmid functions to produce the RNA in prokaryotic cells.
19. A DNA probe or primer comprising 50 or more consecutive
nucleotides from the coding region of SEQ ID NO:3.
20. An RNA probe or primer comprising 50 or more consecutive
nucleotides from SEQ ID NO:4.
21. A probe or primer comprising SEQ ID NO:34 OR SEQ ID NO:35.
Description
BACKGROUND OF THE INVENTION
The DNA at the ends or telomeres of the chromosomes of eukaryotes
usually consists of tandemly repeated simple sequences. Telomerase
is a ribonucleoprotein enzyme that synthesizes one strand of the
telomeric DNA using as a template a sequence contained within the
RNA component of the enzyme. See Blackburn, E. H. (1992) Annu. Rev.
Biochem. 61:113-129, incorporated herein by reference.
The RNA component of a mammalian telomerase has not been reported
in the scientific literature to date, although, human and mouse
telomerases are known to synthesize telomeric repeat units with the
sequence 5'-TTAGGG-3'. See Morin, G. B. (1989) Cell 59:521-529;
Morin, G. B. (1991) Nature 353:454-456; Prowse, et al. (1993) PNAS
90:1493-1497, incorporated herein by reference. This knowledge has
not been sufficient to enable the isolation and identification of
the remainder of the nucleotide sequence of the RNA component of
either of these telomerases. The RNA component of the telomerase
enzymes of Saccharomyces cerevisiae, certain species of
Tetrahymena, as well as that of other ciliates, such as Oxytricha,
Euplotes and Glaucoma, has been sequenced and reported in the
scientific literature. See Singer, M. S. and D. E. Gottschling
(1994) Science 266:404-409; Lingner et al. (1994) Genes &
Development 8:1984-1988; Greider, C. W. and E. H. Blackburn (1989)
Nature 337:331-337; Romero, D. P. and E. H. Blackburn (1991) Cell
67:343-353; Shippen-Lentz, D. and E. H. Blackburn (1990) Science
247:546-552. The teachings of each of these references are
incorporated herein by reference. The telomerase enzymes of these
ciliates synthesize telomeric repeat units distinct from that in
mammals.
There is a great need for more information about mammalian
telomerase. Despite the seemingly simple nature of the repeat units
of telomeric DNA, scientists have long known that telomeres have an
important biological role in maintaining chromosome structure and
function. More recently, scientists have speculated that loss of
telomeric DNA may act as a trigger of cellular senescence and aging
and that regulation of telomerase may have important biological
implications. See Greider, C. W. (1994) Curr. Opin. Genetics Devel.
4:203-211; Harley, C. B. (1991) Mutation Res. 256:271-282; Harley,
C. B. et al. (1990) Nature 345:458-460, incorporated herein by
reference.
Methods for detecting telomerase activity, as well as for
identifying compounds that regulate or affect telomerase activity,
together with methods for therapy and diagnosis of cellular
senescence and immortalization by controlling telomere length and
telomerase activity, have also been described. See PCT patent
publication No. 93/23572, published Nov. 25, 1993, incorporated
herein by reference.
Significant improvements to and new opportunities for
telomerase-mediated therapies and telomerase assays and screening
methods could be realized if nucleic acids comprising the RNA
component and/or encoding the protein components of telomerase were
available in pure or isolated form and the nucleotide sequences of
such nucleic acids were known.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides the RNA component
of, as well as the gene for the RNA component of, human telomerase
in substantially pure form, as well as nucleic acids comprising all
or at least a portion of the nucleotide sequence of the RNA
component of human telomerase (hTR). The present invention also
provides the RNA component of, and the gene for the RNA component
of, mouse telomerase in substantially pure form, as well as nucleic
acids comprising all or at least a portion of the nucleotide
sequence of the RNA component of mouse telomerase (mTR). The
present invention further provides RNA component nucleic acids and
genes encoding such RNA component nucleic acids or portions
thereof, from other species, including the RNA component of rat
(rTR), Chinese hamster (cTR) and bovine telomerase (bTR). The RNA
components include, but are not limited to, the RNA components of
mammals, such as primates. Other nucleic acids of the invention
include nucleic acids with sequences complementary to the RNA
component; nucleic acids with conserved nucleotide residues of RNA
components; nucleic acids with sequences related to, but distinct
from, nucleotide sequences of the RNA components and which interact
with the RNA component or the gene for the RNA component of the
protein components of human telomerase in a useful way; and nucleic
acids that do not share significant sequence homology or
complementarity to the RNA component or the gene for the RNA
component but act on the RNA component in a desired and useful way.
As described more fully below, the nucleic acids of the invention
include both DNA and RNA molecules and modified analogues of either
and serve a variety of purposes.
Thus, one type of nucleic acid of the invention is an antisense
oligonucleotide that can be used in vivo or in vitro to inhibit the
activity of a mammalian telomerase, such as human telomerase. Such
oligonucleotides can block telomerase activity in a number of ways,
including by preventing transcription of the telomerase gene (for
instance, by triple helix formation) or by binding to the RNA
component of telomerase in a manner that prevents a functional
ribonucleoprotein telomerase from assembling or prevents the RNA
component, once assembled into the telomerase enzyme complex, from
serving as a template for telomeric DNA synthesis. Typically, and
depending on mode of action, these oligonucleotides of the
invention comprise a specific sequence of from about 10 to about 25
to 200 or more nucleotides that is either identical or
complementary to a specific sequence of nucleotides in the RNA
component of telomerase or the gene for the RNA component of
telomerase.
Another type of nucleic acid of the invention is a ribozyme able to
cleave specifically the RNA component of a mammalian telomerase,
rendering the enzyme inactive. Yet another type of nucleic acid of
the invention is a probe or primer that binds specifically to the
RNA component of a mammalian telomerase and so can be used, e.g.,
to detect the presence of telomerase in a sample. Finally, nucleic
acids of the invention include recombinant expression plasmids for
producing the nucleic acids of the invention. One type of such a
plasmid is a plasmid used for human gene therapy or for use in
mouse, rat, Chinese hamster and bovine or other mammalian
experimental models. Plasmids of the invention for human gene
therapy or mammalian models come in a variety of types, including
not only those that encode antisense oligonucleotides or ribozymes,
but also those that drive expression of the RNA components of
mammalian telomerase or a deleted or otherwise altered (mutated)
version of the RNA component of human, mouse, rat, Chinese hamster
or bovine (or other species with RNA component sequences homologous
to these RNA components and/or which encode a functional RNA
component when combined with the appropriate telomerase protein
component) telomerase or the gene for the same.
In a second aspect, the invention provides methods for treating a
condition associated with the telomerase activity within a cell or
group of cells by contacting the cell(s) with a therapeutically
effective amount of an agent that alters telomerase activity in
that cell. Such agents include the telomerase RNA
component-encoding nucleic acids, triple helix-forming
oligonucleotides, antisense oligonucleotides, ribozymes, and
plasmids for human gene therapy or mammalian models described
above. In a related aspect, the invention provides pharmaceutical
compositions comprising these therapeutic agents together with a
pharmaceutically acceptable carrier or salt.
In a third aspect, the invention provides diagnostic methods for
determining the level, amount, or presence of the RNA component of
human telomerase, mouse telomerase, telomerase, or telomerase
activity in a cell, cell population, or tissue sample, or an
extract of any of the foregoing. In a related aspect, the present
invention provides reagents for such methods (including the primers
and probes noted above), optionally packaged into kit form together
with instructions for using the kit to practice the diagnostic
method.
In a fourth aspect, the present invention provides recombinant
telomerase preparations and methods for producing such
preparations. Thus, the present invention provides a recombinant
human, mouse, rat, Chinese hamster, bovine or other mammalian
telomerase that comprises the protein components of these
telomerases as well as the protein components of telomerase from
another mammalian species with an RNA component substantially
homologous to the RNA component of human, mouse, rat, Chinese
hamster, bovine or other telomerase in association with a
recombinant RNA component of the inventions. Such recombinant RNA
component molecules of the invention include those that differ from
naturally-occurring RNA component molecules by one or more base
substitutions, deletions, or insertions, as well as RNA component
molecules identical to a naturally-occurring RNA component molecule
that are produced in recombinant host cells. The method for
producing such recombinant telomerase molecules comprises
transforming a eukaryotic host cell that expresses the protein
components of telomerase with a recombinant expression vector that
encodes an RNA component molecule of the invention, and culturing
said host cells transformed with said vector under conditions such
that the protein components and RNA component are expressed and
assemble to form an active telomerase molecule capable of adding
sequences (not necessarily the same sequence added by native
telomerase) to telomeres of chromosomal DNA.
In a fifth aspect, the invention provides methods for purifying the
protein components of human telomerase as well as the protein
components of telomerase from another mammalian species with an RNA
component substantially homologous to the RNA component of human
telomerase. The resent invention also provides methods for
isolating and identifying nucleic acids encoding such protein
components. In related aspects, the present invention provides
purified human, mouse, rat, Chinese hamster, or bovine telomerase
and purified telomerase of other mammalian species with an RNA
component substantially homologous to the coding regions of the RNA
components of human, mouse, rat, Chinese hamster, bovine or other
mammalian telomerase, as well as purified nucleic acids that encode
one or more components of such telomerase preparations. The present
invention also provides pharmaceutical compositions comprising as
an active ingredient the protein components of telomerase or a
nucleic acid that encodes or interacts with a nucleic acid that
encodes a protein component of telomerase.
In a sixth aspect, the invention provides a mammalian model system
through which telomere regulation can be studied in vivo. A mouse,
rat or hamster model, particularly a knockout mouse, offers an
excellent system in which the role of telomerase in aging and
immortalization can be directly tested. Altered telomerase and
compounds that affect the telomerase activity can be used to
determine the effects of telomerase on both cell viability and
organismal development.
Other features and advantages of the invention will be apparent
from the following description of the drawings, preferred
embodiments of the invention, the examples, and the claims.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-1B are the DNA sequence (SEQ ID NO:1) encoding the human
telomerase RNA component.
FIG. 2 is the RNA sequence (SEQ ID NO:2) of the human telomerase
RNA component.
FIG. 3 is the DNA sequence (SEQ ID NO:3) encoding the mouse
telomerase RNA component.
FIG. 4 is the RNA sequence (SEQ ID NO:4) of the mouse telomerase
RNA component.
FIG. 5 is a comparison of the mouse and human ribonucleotide
sequences that comprise the RNA component of telomerase. The coding
region for each begins at the +1 position indicated.
FIG. 6 is a diagram of a targeting construct for knocking out the
mouse telomerase RNA component.
FIGS. 7A-7B are a comparison of the DNA sequences encoding human,
mouse, rat (SEQ ID NO:5), Chinese hamster (SEQ ID NO:43) and bovine
(SEQ ID NO:44) telomerase RNA components.
FIGS. 8A-8E show the putative secondary structure folding of human,
mouse, rat, Chinese hamster and bovine telomerase RNA
components.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods, reagents, and
pharmaceutical compositions relating to the ribonucleoprotein
mammalian telomerases. The invention in part arises out of the
cloning and isolation of the RNA components of mammalian
telomerases, particularly human, mouse, rat, Chinese hamster and
bovine telomerases and the genes for those RNA components. The
nucleotide sequences of the RNA components of both human and mouse
telomerase are shown in FIGS. 2 and 4, respectively. The sequences
of the rat, Chinese hamster and bovine telomerase RNA components
are shown in FIGS. 7A-7B. For convenience, the sequences are shown
using the standard abbreviations for nucleotides (A is adenine, G
is guanine, C is cytidine, T is thymine, and U is uridine). The DNA
sequences for the human and mouse telomerase RNA genes are shown in
FIGS. 1A-1B and 3, respectively.
The sequences in FIGS. 2, 4, and 7 are shown in the 5'-3' direction
and are numbered for reference. The template sequence of the human
RNA component (FIG. 2) is believed to be located within the region
defined by nucleotides 50-60 (5'-CUAACCCUAAC-3'), which is
complementary to a telomeric sequence composed of about
one-and-two-thirds telomeric repeat units.
These sequences were derived from cDNA clones and from the genomic
clone of the RNA components. When the RNA component is first
transcribed from the corresponding human or mouse gene, at least
some of the RNA transcripts produced are much longer than the
.about.560 and .about.535 nucleotide sequences shown and in fact
may comprise more than 1000 nucleotides. However, fully functional
telomerase molecules can be assembled from transcripts consisting
of the .about.560 and .about.535 nucleotide sequences shown in the
figures.
The 3'-end of the RNA component in native human telomerase is
believed to lie within the region defined by nucleotides 514-559 in
the human sequence above; one analysis suggests that the 3'-end may
be the U residue at nucleotide 538. Recombinant RNA component
molecules comprising less than nucleotides 1-559 of the human
sequence shown above can also be used to prepare active
telomerase.
THE RNA COMPONENT OF HUMAN TELOMERASE
The cloning of the RNA component of human telomerase required a
novel method involving negative selection and cycles of positive
selection, described below. Initially, however, an attempt was made
to clone the RNA component using reverse transcription and a method
for cloning the ends of cDNA called "5'-RACE PCR amplification".
The reverse transcription reaction was initiated with a primer
identical to the repeat unit in the single-strand portion of human
telomeric DNA and thus complementary to a sequence believed to be
present in the RNA component of human telomerase. The primer also
comprised, at its 5'-end, a sequence corresponding to a restriction
enzyme recognition site. However, when the cDNA produced by the
reverse transcription reaction and PCR amplification was examined
by gel electrophoresis and nucleotide sequence analysis of the
bands of nucleic acid present in the gel, only ribosomal RNA
sequences were detected. Similar problems were encountered when
variations of this 5'-RACE approach were attempted using nested
primers.
The successful cloning effort began with the preparation of cDNA
from purified preparations of human telomerase, as well as from
cell lines that have human telomerase activity and from cell lines
that do not have detectable human telomerase activity. The method
used to prepare the cDNA is described in detail in Example 1,
below. Two negative selection steps and successive cycles of
positive selection were used in conjunction with the cDNA
preparations from the two human cell lines to lower the
concentration of unwanted sequences and to raise the concentration
of the desired RNA component sequences.
The negative selection steps involved the preparation of
biotinylated PCR product from cDNA prepared from a human cell line
that does not have detectable telomerase activity. The biotinylated
PCR product was denatured and then rehybridized in a solution
comprising a much lower concentration of non-biotinylated PCR
product (100 biotinylated product:1 non-biotinylated product) from
cDNA prepared from a human cell line that does have telomerase
activity. Given the possibility that the telomerase negative cell
line might contain some low amount of the RNA component, the
hybridization step was conducted to discriminate or select against
only RNA expressed abundantly in both cell lines. After
hybridization to a C.sub.0 t selected to allow hybridization of the
most abundantly expressed RNA, the unwanted material was removed by
binding to streptavidinylated magnetic particles; the supernatant
remaining after particle collection contained the desired cDNA for
the RNA component of human telomerase. The process for PCR
amplification of cDNA is described in Example 2, below.
This material was further enriched for the desired cDNA by
successive cycles of positive selection. In the positive selection
step, a biotinylated probe complementary to the predicted template
sequence in the RNA component of human telomerase was hybridized to
PCR product from an enriched (by negative selection) sample of the
PCR-amplified cDNA from a human cell line that has telomerase
activity. After hybridization, the probe/target complexes were
bound to avidinylated magnetic beads, which were then collected and
used as a source of nucleic acid enriched in RNA component
sequences in further cycles of positive selection. The positive
selection process is described in more detail in Examples 3 and 4,
below.
After the third cycle of positive selection, the amplification
products were separated by gel electrophoresis, and sections of the
gel corresponding to nucleic acids .about.200 bp in size were
removed. The nucleic acids were then eluted from the gel sections
and amplified by PCR. The PCR amplification products were digested
with restriction enzyme Not1 and then inserted by ligation into the
Not1 site of plasmid pBluescriptIISK+, commercially available from
Stratagene. The resulting plasmids were transformed into E. coli
host cells, and individual colonies were isolated and used as a
source of nucleic acid for further analysis and DNA sequencing.
Individual colonies were grown in the wells of a 96-well microtiter
plate, which was then used as a master plate, and blots of DNA from
the colonies in the plate were prepared and hybridized to a probe
comprising a telomeric repeat sequence and therefore complementary
to the RNA component of human telomerase. A number of clones
positive by this test were then analyzed by DNA sequencing and a
variety of other tests.
These other tests included the following: (1) determination of
whether antisense oligonucleotides complementary to the putative
RNA component would inhibit telomerase activity in human cell
extracts known to contain telomerase (Greider, C. W. and E. H.
Blackburn (1989) supra); (2) determination of whether PCR primers
specific for a putative RNA component clone sequence could be used
to amplify a nucleic acid present in a telomerase sample and
whether the product observed, if any, would track telomerase
activity during purification of telomerase (Greider, C. W. and E.
H. Blackburn (1987) supra); and (3) determination of whether PCR
primers specific for a putative RNA component clone sequence could
be used to amplify a nucleic acid present in greater abundance in
cell extracts from cells in which telomerase activity is known to
be high (i.e., tumor cells) than in cell extracts from cells known
to produce no or only low amounts of telomerase activity. One
clone, designated plasmid pGRN7, produced results in these tests
consistent with the determination that the plasmid comprised cDNA
corresponding to the RNA component of human telomerase.
Thus, antisense oligonucleotides corresponding to sequences of the
putative RNA component sequence of pGRN7 exhibited inhibition of
telomerase activity in vitro. Likewise, when telomerase was
purified from cell extracts by a process involving (1) DEAE
chromatography; (2) Sephadex S300 chromatography; and (3) either
glycerol gradient, SP sepharose, or phenyl sepharose separation and
fractions collected, PCR primers specific for the putative RNA
component sequence of pGRN7 amplified a nucleic acid of the
appropriate size, and the amount of amplification product
correlated well with the amount of telomerase activity observed in
the fractions collected. Finally, cell extracts from normal (no
detectable telomerase activity) and cancer (telomerase activity
present), as well as testis (telomerase activity present), cells
showed corresponding amounts of PCR product upon reverse
transcription and PCR amplification (RT-PCR) with primers specific
for the putative RNA component comprised in pGRN7. The protocol for
the RT-PCR is described in Examples 5 and 6, below.
The above results provided convincing evidence that the RNA
component of human telomerase had been cloned. Therefore, plasmid
pGRN7 was then used to isolate a genomic clone for the RNA
component from a human cell line, as described in Example 7, below.
The genomic clone was identified in and isolated from a genomic
library of human DNA inserted into a lambda vector FIXII purchased
from Stratagene. The desired clone comprising the RNA component
gene sequences contained an .about.15 KB insert and was designated
clone 28-1. This clone has been deposited with the American Type
Culture Collection and is available under the ATCC accession No.
75925. Various restriction fragments were subcloned from this phage
and sequenced. The gene has also been localized to the distal end
of the q arm of chromosome 3. The sequence information obtained
from a SauIIIA1 restriction enzyme recognition site at one end of
the .about.15 kb insert to an internal HindIII restriction enzyme
recognition site, which comprises all of the mature RNA component
sequence as well as transcription control elements of the RNA
component gene, of lambda clone 28-1 is shown in FIGS. 1A-1B using
the standard deoxyribonucleotide abbreviations and depicted in the
5'-3' direction.
The RNA component sequence begins at base 1459 and ends at base
2017. A variety of transcription control elements can also be
identified in the sequence. An A/T Box consensus sequence is found
at nucleotides 1438-1444; PSE consensus sequences are found at
nucleotides 1238-1250 as well a nucleotides 1406-1414; a CAAT Box
consensus sequence is found at nucleotides 1399-1406; an SP1
consensus sequence is found at nucleotides 1354-1359; and a
beta-interferon response element consensus sequence is found at
nucleotides 1234-1245.
The plasmids described above that were constructed during the
cloning of the RNA component of human telomerase and the gene for
the RNA component are important aspects of the present invention.
These plasmids can be used to produce the RNA component of, as well
as the gene for, human telomerase in substantially pure form, yet
another important aspect of the present invention. In addition,
those of skill in the art recognize that a variety of other
plasmids, as well as non-plasmid nucleic acids in substantially
pure form, that comprise all or at least a portion of the
nucleotide sequence of the RNA component of human telomerase are
provided by the present invention.
THE RNA COMPONENT OF MOUSE TELOMERASE
The cloning of the mouse RNA component of telomerase was initiated
by isolating highly purified mouse telomerase fractions using five
different column chromatography steps. These active fractions were
highly enriched for the RNA component and provided essential
ribonucleoenzyme materials for cloning procedures. The cloning
procedures are described in detail in Examples 9-13, and the mouse
telomerase RNA component gene is shown in FIG. 3. The RNA component
sequence begins at base 531 and ends at base 1065.
The small RNAs which co-purified with telomerase were sequenced and
those that had the template region CTAACCCTAA were identified and
targeted as potential RNAs involved in telomerase elongation. The
potential RNA generated while cloning the human telomerase RNA
component also showed characteristics of a telomerase component.
Using the genomic clone of the human RNA component, mouse genomic
fragments were identified from a lambda library that hybridized to
a human probe at medium stringency. Approximately five genome
equivalents were screened with a probe that contained 500 bp of the
human telomerase RNA gene. The positive lambda phage were
restriction mapped and four were identical to each other. Two
positive bands in a Pst1 digest of the clones which hybridized to
the human gene were subcloned and sequenced. The sequence was 64%
identical to the 550 nucleotide coding region of the human
telomerase RNA indicating that this clone might be the mouse
telomerase RNA gene. Outside of coding region, the sequence
identity dropped to 45%. The sequence identity in the coding region
of the human and putative mouse RNAs is significantly less than
that found for other small RNA genes between human and mouse. The
U-RNA species range from 100% identity for U6 to 85% identity for
U7. The mouse and human RNase P RNA and MRP RNAs are 86% and 78%
identical respectively. Thus the telomerase RNA is the least well
conserved between human and mouse of any mammalian small functional
RNA yet identified. This low level of sequence conservation among
telomerase RNAs has also been found in the ciliates. Lingner, et
al. (1994) supra; Romero, D. P. and E. H. Blackburn, (1991)
supra.
The template region of the human RNA is not absolutely conserved in
the mouse sequence. There are 11 nucleotides in the potential
template of the human RNA CUAACCCUAAC while there are only 9
possible nucleotides in the mouse RNA CCUAACCCU. This change may be
the cause of the decreased processivity of the mouse enzyme
relative to the human enzyme. Prowse, et al. (1993) supra.
Due to the low level of sequence conservation between the human and
mouse clones, several approaches were used to determine whether the
mouse sequence represents the functional telomerase RNA. First,
genomic Southern blots probed at high stringency identified a
single band when probed with the potential coding region (data not
shown). At lower stringency, several bands were observed;
therefore, tests were done to determine if the sequence obtained
was expressed as functional RNA. RT-PCR from total RNA was
performed with or without the initial reverse transcriptase step.
Using two primers for the initial amplification, followed by a
second round of PCR with an internal `nested` primer, one band with
the expected size of 300 bp was amplified. This band was dependent
on the initial reverse transcriptase step indicating it was
generated from RNA and not from low levels of contaminating genomic
DNA. The sequence of this amplified product matched the sequence of
the initial genomic DNA exactly, including in the region of the
CCTAACCCT template.
Functional tests indicated that the cloned sequence represented the
mouse telomerase RNA component. Investigations of co-purification
with telomerase activity (Greider, C. W. and E. H. Blackburn (1987)
supra) and for antisense oligonucleotide inhibition (Greider, C. W.
and E. H. Blackburn (1989) supra) showed positive results.
Telomerase was purified through chromatography over DEAE agarose,
spermine agarose and phenyl sepharose; the telomerase activity was
assayed, and RNA was prepared from all of the fractions. Northern
analysis showed an RNA of approximately 550 bp was present in the
active fractions of these columns. The RNA also co-purified with
telomerase activity over a glycerol gradient. The size of the RNA
in mouse cells is similar to that found in human cells.
(Villeponteau, et al. (1995) in press).
Previous work with Tetrahymena telomerase showed that antisense
oligonucleotides that cover the template inhibit telomerase and
that oligonucleotides with 3' ends adjacent to the template are
elongated by telomerase (Greider, C. W. and E. H. Blackburn (1989)
supra; Lingner, et al. (1994) supra). Oligonucleotides directed
against the mouse RNA were examined for their effects on both
inhibition and elongation of the mouse RNA. Oligonucleotides
complementary to the candidate mouse telomerase RNA either covering
the template region, MI-2, or hybridizing just 3' to the template
MP-1, were tested for their ability to inhibit telomerase or serve
as primers. For inhibition assays, each oligonucleotide was
pre-incubated with mouse telomerase before the substrate
d(TTAGGG).sub.3 was added. MI-2 which covers 11 nucleotides 3' of
the template was an efficient inhibitor of telomerase activity at
both 4 .mu.M or 10 .mu.M. Incubation with MP-1 or two other
oligonucleotides that hybridize 3' of the template, MP-2 and MP-3,
did not inhibit elongation, indicating that oligonucleotides that
do not cross the template do not block elongation.
To determine the ability of the antisense oligonucleotides to serve
as substrates, each oligonucleotide was added to a telomerase
reaction in the absence of (TTAGGG).sub.3 primer. The
oligonucleotide MP-1, whose 3' end is just adjacent to the 3' RNA
template, was elongated by the addition of 8 residues and the
addition of these products was RNase sensitive. Two control
oligonucleotides, MP-2 and MP-3, were not elongated in this
experiment. The addition of 8 nucleotides to MP-1 is consistent
with the addition of the template complementary sequence AGGGTTAG
onto the primer (see below).
To examine the specificity of the antisense primer inhibition and
elongation, three control oligonucleotides (MI-3, MI-4 and MI-5)
were synthesized. MI-4 extended RNA complementarity of MP-1
oligonucleotide through the template region of the RNA. This new
oligonucleotide inhibited d(TTAGGG).sub.3 elongation so that it no
longer served as a substrate for elongation although it has
telomeric sequence at the 3' end.
To determine the specificity of MI-2 inhibition, the sequence of
the 5' most 9 nucleotides of the oligonucleotide was changed so
that it was no longer complementary to the RNA. This new
oligonucleotide, MI-3, no longer inhibited d(TTAGGG).sub.3
elongation; however, it did serve as a substrate for elongation due
to the 3' telomeric repeats. The predominant product migrated in
the gel at position primer +5 as predicted since the 3' end of the
oligonucleotide had the sequence TTAGG. Telomerase is expected to
synthesize the five nucleotides GTTAG onto a primer with this 3'
sequence.
As a last control, the 3' most 9 residues of the inhibitory MI-2
oligonucleotide were changed so that the resulting oligonucleotide
(designated MI-5), would no longer hybridize to the CCUAACCCU
template. MI-5 was not a telomerase substrate and did not inhibit
d(TTAGGG).sub.3 elongation. Thus removing the complementarity of
the antisense oligonucleotides abolished their ability to interact
with telomerase in a predictable manner. To determine the
inhibition efficiency of MI-2 and MI-4, telomerase was
pre-incubated with a decreasing concentration of each antisense
oligonucleotide prior to the addition of d(TTAGGG).sub.3, and then
tested for elongation. Inhibition was complete at 0.2 .mu.M for
both oligonucleotides. These data show that the cloned RNA gene is
a functional component of mouse telomerase.
Both mouse and Xenopus telomerase RNAs generate predominately one
band during elongation. The position of the dissociation has been
mapped to the first G residue in the sequence TTAG (Mantell, L. L.
and C. W. Greider (1994) EMBO J. 13:1211-3217; Prowse, et al.
(1993) supra). To determine the sequence added onto the MP-1
oligonucleotide, dideoxyTTP was substituted for the deoxyTTP in the
elongation reaction. Telomerase reactions were carried out in the
presence of .sup.32 P-dGTP, dATP and dTPP or .sup.32 P-dGTP, dATP
and ddTPP. Using the primer (TTAGGG).sub.3 in the presence of dTTP,
the typical predominant band at primer +4 was observed (Prowse, et
al. (1993) supra). When ddTTP was substituted, all incorporation
was abolished due to the incorporation of dTMP before the addition
of labeled .sup.32 P-dGTP. Therefore, no labeled products are
generated. Using MP-1, the 8 nucleotide labeled product observed
with dTTP was reduced to 5 nucleotides in size when ddTTP was
added. Again, this is consistent with the synthesis of AGGGTTAG
onto the 3' end of the primer; chain termination with ddT inhibited
the addition of the last TAG sequence.
If the entire potential template region of the mouse RNA,
CCUAACCCU, were copied, and primer translocation occurred at the
end of the template, a pause at the second G residue in the repeat
d(TTAGGG) would be expected. Lingner, et al. (1994) supra; Greider,
C. W. and E. H. Blackburn (1989) supra. Using permuted sequence
primer oligonucleotides, both the human and the mouse telomerase
enzymes pause or dissociate at the first G in the sequence. Prowse,
et al. (1993) supra; Morin, G. B. (1989) supra. These data and the
elongation of the antisense oligonucleotide describe above, show
that the 5' most C which is present in the mouse but not the human
template region does not serve as a template in the mouse RNA
component. This is similar to the 5' most C in the Stylonichia
telomerase RNA template which is apparently also not used as a
template position (Lingner, et al. (1994) supra). Similarly, when
an additional C residue was added to the 5' most border of the
Tetrahymena template region, this extra C residue was not
incorporated into reaction products (Autexier, C. and C. W. Greider
(1994) unpublished data) indicating that there is an active
mechanism to determine the boundaries of the template sequence
within the telomerase RNA.
Primer extension and RT-PCR were used to characterize the 5' end of
the mouse RNA. Northern analysis showed that the mouse and human
telomerase RNAs are similar in size. Primer extension from total
mouse RNA generated two distinct bands, one that mapped 20
nucleotides longer than the human 5' end and one that mapped 15
nucleotides shorter. To determine whether the RNA extends past the
position of the mapped human 5' end, an oligonucleotide to this
sequence was used in an RT-PCR reaction. Total RNA was reverse
transcribed using random hexamers as primers. This cDNA was then
amplified using an internal primer and an 18 nucleotide primer
which would hybridize to the 5' most region identified by primer
extension. A unique band of 450 nucleotides was generated and this
product was further amplified with an internal nested primer, again
the correct sized product of 200 nucleotides was generated. The
products were cloned and the sequence corresponded to the genomic
sequence shown in FIG. 4. This indicates that the RNA starts at the
sequence 5' CUCGACC which is designated as +1 in the mouse sequence
of FIG. 5. It is not yet determined whether the difference in start
sites for the human and mouse telomerase RNAs is of functional
significance. In FIG. 5, the conserved ribonucleotide residues
between the human and mouse RNA sequences are boxed, and the
template regions are boxed and highlighted in grey.
The expression of the RNA was assayed in cell lines and tissues
where telomerase activity levels vary. Primary Mus spretus
fibroblasts lack detectable telomerase activity and show telomere
shortening similar to that found in human cells, while immortalized
fibroblasts have telomerase activity. Northern analysis showed that
immortalized fibroblasts expressed the 550 nucleotide RNA while it
was not detected in primary cells with no telomerase activity.
Similarly, the level of RNA present in mouse tissues paralleled the
level of telomerase activity. RNA was detected in testes and liver
both of which have high levels of telomerase activity. In addition,
the RNA level was high in spleen. Telomerase activity is present in
spleen although the relative level of activity compared to liver
and testes is not known due to the presence of inhibitors in the
crude spleen extracts.
As a general point regarding the nucleic acids and preparations
containing the same of the invention, those of skill in the art
recognize that the nucleic acids of the invention include both DNA
and RNA molecules, as well as synthetic, non-naturally occurring
analogues of the same, and heteropolymers of deoxyribonucleotides,
ribonucleotides, and/or analogues of either. The particular
composition of a nucleic acid or nucleic acid analogue of the
invention will depend upon the purpose for which the material will
be used and the environment(s) in which the material will be
placed. Modified or synthetic, non-naturally occurring nucleotides,
have been designed to serve a variety of purposes and to remain
stable in a variety of environments, such as those in which
nucleases are present, as is well known in the art. Modified or
synthetic non-naturally occurring nucleotides, as compared to the
naturally occurring nucleotides, as compared to the naturally
occurring ribo- or deoxyribonucleotides, may differ with respect to
the carbohydrate (sugar), phosphate linkage, or base portions, of
the nucleotide, or may even contain a non-nucleotide base (or no
base at all) in some cases. See, e.g., Arnold et al., PCT Patent
Publication No. WO 89/02439, entitled "Non-nucleotide Linking
Reagents for Nucleotide Probes" incorporated herein by
reference.
Just as the nucleic acids of the invention can comprise a wide
variety of nucleotides, so too can those nucleic acids serve a wide
variety of useful functions. RNA probes or primers, especially
those comprising a consecutive sequence of about 20 to 200 or more
ribonucleotides encompassing conserved sequences between SEQ ID
NO:1 and SEQ ID NO:3 are useful to detect telomerase RNA components
in other species, and to inhibit or enhance telomerase activity as
described below. DNA probes or primers encoding a consecutive
sequence of about 20 to 200 or more ribonucleotides encompassing
conserved sequences between SEQ ID NO:1 and SEQ ID NO:3, as well as
DNA sequences that hybridize under moderately stringent conditions
(Ausubel, et al. (1989) Current Protocols in Molecular Biology,
John Wiley & Sons, NY) to these conserved sequences may also be
used for identification, diagnostic, assay and therapeutic
purposes. In particular, both RNA and DNA probes may be used to
identify telomerases and telomerase activity in other mammalian
cells. RNA sequences that are substantially homologous to the
conserved sequences between SEQ ID NO:1 and SEQ ID NO:3, or that
hybridize to these conserved sequences under moderately stringent
conditions are also within the scope of this invention.
Another especially useful type of nucleic acid of the invention is
an antisense oligonucleotide that can be used in vivo or in vitro
to inhibit the activity of human or another mammalian telomerase.
Antisense oligonucleotides comprise a specific sequence of from
about 10 to about 25 to 200 or more (i.e., large enough to form a
stable duplex but small enough, depending on the mode of delivery,
to administer in vivo, if desired) nucleotides complementary to a
specific sequence of nucleotides in the RNA component of a
mammalian telomerase. The mechanism of action of such
oligonucleotides can involve binding of the RNA component either to
prevent assembly of the functional ribonucleoprotein telomerase or
to prevent the RNA component from serving as a template for
telomeric DNA synthesis.
Illustrative antisense oligonucleotides of the invention that serve
to inhibit telomerase activity in vivo and/or in vitro include the
oligonucleotides mentioned above in connection with the tests to
determine whether clone pGRN7 comprised the cDNA for the RNA
component of human telomerase. Three such oligonucleotides were
synthesized as 2'-O-methyl RNA olignucleotides and are more
resistant to hydrolysis than unmodified RNA oligonucleotides, and,
as noted above, were used to demonstrate inhibition of telomerase
activity in vitro. The sequence of each of these O-methyl RNA
oligonucleotides is shown below.
T3 5'-CUCAGUUAGGGUUAGACAAA-3' (SEQ ID NO:6)
P3 5'-CGCCCUUCUCAGUUAGGGUUAG-3' (SEQ ID NO:7)
TA3 5'-GGCGCCUACGCCCUUCUCAGUU-3' (SEQ ID NO:8)
These oligonucleotides can also be used to inhibit telomerase
activity in human cells.
Those of skill in the art will recognize that the present invention
provides a wide variety of antisense oligonucleotides able to
inhibit telomerase activity. Another useful antisense
oligonucleotide of the invention is oligonucleotide Tel-AU, which
has the sequence 5'-CAGGCCCACCCTCCGCAACC-3' (SEQ ID NO:9), and
which, like any of the antisense oligonucleotides of the invention,
can be synthesized using phosphorothioate nucleotides, chiralmethyl
phosphonates, naturally occurring nucleotides, or mixtures of the
same to impart stability and the desired T.sub.m. Those of skill in
the art recognize that a wide variety of modified nucleotide
analogues, such as O-methyl ribonucleotides, phosphorothioate
nucleotides, and methyl phosphonate nucleotides, can be used to
produce nucleic acids of the invention with more desired properties
(i.e., nuclease-resistant, tighter-binding, etc.) than those
produced using naturally occurring nucleotides. Other techniques
for rendering oligonucleotides nuclease-resistant include those
described in PCT patent publication No. 94/12633.
In addition, to the antisense oligonucleotides of the invention,
one can construct oligonucleotides that will bind to duplex nucleic
acid either in the folded RNA component or in the gene for the RNA
component, forming a triple helix-containing or triplex nucleic
acid to inhibit telomerase activity. Such oligonucleotides of the
invention are constructed using the base-pairing rules of triple
helix formation and the nucleotide sequence of the RNA component.
Such oligonucleotides can block telomerase activity in a number of
ways, including by preventing transcription of the telomerase gene
or by binding to a duplex region of the RNA component of telomerase
in a manner that prevents the RNA component either from forming a
functional ribonucleoprotein telomerase or from serving as a
template for telomeric DNA synthesis. Typically, and depending on
mode of action, the triplex-forming oligonucleotides of the
invention comprise a specific sequence of from about 10 to about 25
to 200 or more (i.e., large enough to form a stable triple helix
but small enough, depending on the mode of delivery, to administer
in vivo, if desired) nucleotides "complementary" (in this context,
complementary means able to form a stable triple helix) to a
specific sequence in the RNA component of telomerase or the gene
for the RNA component of telomerase.
In addition to the antisense and triple helix-forming
oligonucleotides of the invention, "sense" oligonucleotides
identical in sequence to at least a portion of the RNA component of
human telomerase or another mammalian telomerase can also be used
to inhibit telomerase activity. Oligonucleotides of the invention
of this type are characterized in comprising either (1) less than
the complete sequence of the RNA component needed to form a
functional telomerase enzyme or (2) the complete sequence of the
RNA component needed to form a functional telomerase enzyme as well
as a substitution or insertion of one or more nucleotides that
render the resulting RNA non-functional. In both cases, inhibition
of telomerase activity is observed due to the "mutant" RNA
component binding the protein components of the telomerase to form
an inactive telomerase molecule. The mechanism of action of such
oligonucleotides thus involves the assembly of a non-functional
ribonucleoprotein telomerase or the prevention of assembly of a
functional ribonucleoprotein telomerase. Sense oligonucleotides of
the invention of this type typically comprise a specific sequence
of from about 20, 50, 200, 400, 500, or more nucleotides identical
to a specific sequence of nucleotides in the RNA component of a
telomerase.
Thus, another oligonucleotide of the invention comprises an altered
or mutated sequence of the RNA component of human or another
mammalian telomerase. Yu, et al. (1990) Nature 344:126, shows that
a mutated form of the RNA component of Tetrahymena telomerase can
be incorporated into the telomerase of Tetrahymena cells and that
the incorporation has deleterious effects on those cells.
Incorporation of mutated forms of the RNA component of human or
another mammalian telomerase may have similar effects on human or
mammalian cells that otherwise have telomerase activity without
affecting normal human cells that do not have telomerase activity.
Such mutated forms include those in which the sequence
5'-CTAACCCTA-3' is mutated to 5'-CAAACCCAA-3', 5'-CCAACCCCAA-3', or
5'-CTCACCCTCA-3'. Each of these altered RNA component sequences
alters the telomeric repeat units incorporated into the chromosomal
DNA, thus affecting chromosome structure and function. Such
oligonucleotides can be designed to contain restriction enzyme
recognition sites useful in diagnostic methods for the presence of
the altered RNA component via restriction enzyme digestion of
telomeric DNA or an extended telomerase substrate.
To illustrate this aspect of the invention, site-specific
mutagenesis was carried out using a plasmid (designated pGRN33,
available from the American Type Culture Collection under ATCC
accession No. 75926) that comprises an .about.2.5 kb HindIII-SacI
fragment from lambda clone 28-1 (see Example 7, below) as well as
the SV40 origin of replication (but no promoter activity). The
resulting plasmids, designated pGRN34 (comprising 5'-CAACCCAA-3'),
pGRN36 (comprising 5'-CCAACCCCAA-3'), and pGRN37 (comprising
5'-CTCACCCTCA-3'), were transformed into eukaryotic host cells (a
293-derived cell line expressing SV40 large T antigen), and
telomerase assays were conducted using cell extracts from the
transformants.
The assays showed that the telomerase activity in the cells
resulted in the formation of nucleic acids comprising the altered
sequences, indicating that the genomic clone comprised a functional
RNA component gene and that the plasmids comprised an altered but
functional RNA component gene. These results illustrate how the
present invention provides recombinant telomerase preparations and
methods for producing such preparations. The present invention
provides a recombinant human or mammalian telomerase in functional
association with a recombinant RNA component of the invention. Such
recombinant RNA component molecules of the invention include those
that differ from naturally occurring RNA component molecules by one
or more base substitutions, deletions, or insertions, as well as
RNA component molecules identical to a naturally occurring RNA
component molecule that are produced in recombinant host cells. The
method for producing such recombinant telomerase molecules
comprises transforming a eukaryotic host cell that expresses the
protein components of telomerase with a recombinant expression
vector under conditions such that the protein components and RNA
components are expressed and assemble to form an active telomerase
molecule capable of adding sequences (not necessarily the same
sequence added by native telomerase) to telomeres of chromosomal
DNA. Other useful embodiments of such recombinant DNA expression
vectors (or plasmids) include plasmids that comprise the gene for
the RNA component of human telomerase with a deletion, insertion,
or other modification that renders the gene non-functional. Such
plasmids are especially useful for gene therapy to "knock-out" the
endogenous RNA component gene, although a highly efficient
transformation and recombination system is required, to render the
treated cells irreversibly mortal.
Other oligonucleotides of the invention called "ribozymes" can also
be used to inhibit telomerase activity. Unlike the antisense and
other oligonucleotides described above, which bind to an RNA, a
DNA, or a telomerase protein component, a ribozyme not only binds
but also specifically cleaves and thereby potentially inactivates a
target RNA, such as the RNA component of human telomerase. Such a
ribozyme can comprise 5'- and 3'-terminal sequences complementary
to the telomerase RNA. Depending on the site of cleavage, a
ribozyme can render the telomerase enzyme inactive. See PCT patent
publication No. 93/23572, supra. Those in the art upon review of
the RNA sequence of the human or mouse telomerase RNA component
will note that several useful ribozyme target sites are present and
susceptible to cleavage by, for example, a hammerhead motif
ribozyme. Illustrative human ribozymes of the invention of this
type include the ribozymes below, which are RNA molecules having
the sequences indicated:
1: 5'-UAGGGUUACUGAUGAGUCCGUGAGGACGAAACAAAAAAU-3' (SEQ ID NO:10)
2: 5'-UUAGGGUCUGAUGAGUCCGUGAGGACGAAAGACAAAA-3' (SEQ ID NO:11)
3: 5'-UCUCAGUCUGAUGAGUCCGUGAGGACGAAAGGGUUA-3' (SEQ ID NO:12)
4: 5'-CCCGAGACUGAUGAGUCCGUGAGGACGAAACCCGCG-3' (SEQ ID NO:13).
Other optimum target sites for ribozyme-mediated inhibition of
telomerase activity can be determined as described by Sullivan, et
al., PCT patent publication No. 94/02595 and Draper, et al., PCT
publication No. 93/23569, both incorporated herein by reference. As
described by Hu, et al., PCT patent publication No. 94/03596,
incorporated herein by reference, antisense and ribozyme functions
can be combined in a single oligonucleotide. Moreover, ribozymes
can comprise one or more modified nucleotides or modified linkages
between nucleotides, as described above in conjunction with the
description of illustrative antisense oligonucleotides of the
invention.
Thus, the invention provides a wide variety of oligonucleotides to
inhibit telomerase activity. Such oligonucleotides can be used in
the therapeutic methods of the invention for treating disease,
which methods comprise administering to a patient a therapeutically
effective dose of a telomerase inhibitor or activator of the
invention. One can measure telomerase inhibition or activation to
determine the amount of an agent that should be delivered in a
therapeutically effective dose using the assay protocols described
in the copending U.S. patent applications and PCT patent
publication No. 93/23572 noted above. As noted in those application
and discussed above, inhibition of telomerase activity renders an
immortal cell mortal, while activation of telomerase activity can
increase the replicative lifespan of a cell. Telomerase inhibition
therapy is an effective treatment against cancers involving the
uncontrolled growth of immortal cells, and telomerase activation is
an effective treatment to prevent cell senescence. Delivery of
agents that inhibit or block telomerase activity, such as an
antisense oligonucleotide, a triple helix-forming oligonucleotide,
a ribozyme, or a plasmid that drives expression of a mutant RNA
component of telomerase can prevent telomerase action and
ultimately leads to cell senescence and cell death of treated
cells.
In addition, the present invention provides therapeutic methods
that ensure that normal cells remain mortal; for instance, the RNA
component can be modified using standard genetic engineering
procedures to delete all or a portion of a natural gene encoding
the component (e.g., by in vitro mutagenesis) by genetic
recombination. Such cells will then be irreversibly mortal. This
procedure is useful in gene therapy, where normal cells modified to
contain expression plasmids are introduced into a patient, and one
wants to ensure cancerous cells are not introduced or, if such
cells are introduced, then those cells have been rendered
irreversibly mortal.
Because telomerase is active only in tumor, germline, and certain
stem cells of the hematopoietic system in humans, other normal
cells are not affected by telomerase inhibition therapy. Steps can
also be taken to avoid contact of the telomerase inhibitor with
germline or stem cells, although this may not be essential. For
instance, because germline cells express telomerase activity,
inhibition of telomerase may negatively impact spermatogenesis and
sperm viability, suggesting that telomerase inhibitors may be
effective contraceptives or sterilization agents. This
contraceptive effect may not be desired, however by a patient
receiving a telomerase inhibitor of the invention for treatment of
cancer. In such cases, one can deliver a telomerase inhibitor of
the invention in a manner that ensures the inhibitor will only be
produced during the period of therapy, such that the negative
impact on germline cells is only transient.
Other therapeutic methods of the invention employ the telomerase
RNA nucleic acid of the invention to stimulate telomerase activity
and to extend replicative cell life span. These methods can be
carried out by delivering to a cell a functional recombinant
telomerase ribonucleoprotein of the invention to the cell. For
instance, the ribonucleoprotein can be delivered to a cell in a
liposome, or the gene for the RNA component of human telomerase (or
a recombinant gene with different regulatory elements) can be used
in a eukaryotic expression plasmid (with or without sequences
coding for the expression of the protein components of telomerase)
to activate telomerase activity in various normal human cells that
otherwise lack detectable telomerase activity due to low levels of
expression of the RNA component or a protein component of
telomerase. If the telomerase RNA component is not sufficient to
stimulate telomerase activity, then the RNA component can be
transfected along with genes expressing the protein components of
telomerase to stimulate telomerase activity. Thus, the invention
provides methods for treating a condition associated with the
telomerase activity within a cell or group of cells by contacting
the cell(s) with a therapeutically effective amount of an agent
that alters telomerase activity in that cell.
Cells that incorporate extra copies of the telomerase RNA gene can
exhibit an increase in telomerase activity and an associated
extended replicative life span. Such therapy can be carried out ex
vivo on cells for subsequent introduction into a host or can be
carried out in vivo. The advantages of stabilizing or increasing
telomere length by adding exogenous telomerase genes ex vivo to
normal diploid cells include: telomere stabilization can arrest
cellular senescence and allow potentially unlimited amplification
of the cells; and normal diploid cells with an extended life span
can be cultured in vitro for drug testing, virus manufacture, or
other useful purposes. Moreover, ex vivo amplified stem cells of
various types can be used in cell therapy for particular diseases,
as noted above.
Telomere stabilization can also suppress cancer incidence in
replicating cells by preventing telomeres from becoming critically
short as cells near crisis. During crisis, massive genomic
instability is generated as the protective effect of the telomeric
cap is lost. The "genetic deck" is reshuffled, and almost all cells
die. The rare cells that emerge from this process are typically
aneuploid with many gene rearrangements and end up reestablishing
stability in their telomeres by expressing telomerase. If crisis
can be prevented by keeping telomeres long, then the genomic
instability associated with crisis can also be prevented, limiting
the chances than an individual cell will suffer the required number
of genetic mutations needed to spawn a metatastic cancer.
Cells that can be targeted for telomerase gene therapy (therapy
involving increasing the telomerase activity of a target cell) in
humans include but are not limited to hematopoietic stem cells
(AIDS and post-chemotherapy), vascular endothelial cells (cardiac
and cerebral vascular disease), skin fibroblasts and basal skin
keratinocytes (wound healing and burns), chondrocytes (arthritis),
brain astrocytes and microglial cells (Alzheimer's Disease),
osteoblasts (osteoporosis), retinal cells (eye diseases), and
pancreatic islet cells (Type I diabetes).
Typically, the therapeutic methods of the invention involve the
administration of an oligonucleotide or drug that functions to
inhibit or stimulate telomerase activity under in vivo
physiological conditions and will be stable under those conditions.
As noted above, modified nucleic acids may be useful in imparting
such stability, as well as for ensuring delivery of the
oligonucleotide to the desired tissue, organ, or cell. Methods
useful for delivery of oligonucleotides for therapeutic purposes
are described in Inouye et al., U.S. Pat. No. 5,272,065,
incorporated herein by reference.
While oligonucleotides can be delivered directly as a drug in a
suitable pharmaceutical formulation, one can also deliver
oligonucleotides using gene therapy and recombinant DNA expression
plasmids of the invention. One such illustrative plasmid is
described in Example 8, below. In general, such plasmids will
comprise a promoter and, optionally, an enhancer (separate from any
contained within the promoter sequences) that serve to drive
transcription of an oligoribonucleotide, as well as other
regulatory elements that provide for episomal maintenance or
chromosomal integration and for high-level transcription, if
desired. Adenovirus-based vectors are often used for gene therapy
and are suitable for use in conjunction with the reagents and
methods of the present invention. See PCT patent publication Nos.
94/12650; 94/12649; and 94/12629. Useful promoters for such
purposes include the metallothionein promoter, the constitutive
adenovirus major late promoter, the dexamethasone-inducible MMTV
promoter, the SV40 promoter, the MRP polIII promoter, the
constitutive MPSV promoter, the tetracycline-inducible CMV promoter
(such as the human immediate-early CMV promoter), and the
constitutive CMV promoter. A plasmid useful for gene therapy can
comprise other functional elements, such as selectable markers,
identification regions, and other genes. Recombinant DNA expression
plasmids can also be used to prepare the oligonucleotides of the
invention for delivery by means other than by gene therapy,
although it may be more economical to make short oligonucleotides
by in vitro chemical synthesis.
In related aspects, the invention features pharmaceutical
compositions including a therapeutically effective amount of a
telomerase inhibitor or telomerase activator of the invention.
Pharmaceutical compositions of telomerase inhibitors of the
invention include a mutant RNA component of human or another
mammalian telomerase, an antisense oligonucleotide or triple
helix-forming oligonucleotide that binds the RNA component or the
gene for the same of human or another mammalian telomerase, or a
ribozyme able to cleave the RNA component of human or another
mammalian telomerase, or combinations of the same or other
pharmaceuticals in a pharmaceutically acceptable carrier or salt.
Other pharmaceutical compositions of the invention comprise a
telomerase activator preparation, such as purified human telomerase
or mRNA for the protein components of telomerase and the RNA
component of telomerase, and are used to treat senescence-related
disease. The therapeutic agent can be provided in a formulation
suitable for parenteral, nasal, oral, or other mode of
administration. See PCT patent publication No. 93/23572, supra.
The present invention provides diagnostic methods and reagents in
addition to the pharmaceutical formulations and therapeutic methods
described above. The invention provides diagnostic methods for
determining the level, amount, or presence of the RNA component of
telomerase, telomerase, or telomerase activity in a cell, cell
population, or tissue sample. In a related aspect, the present
invention provides useful reagents for such methods, optionally
packaged into kit form together with instruction for using the kit
to practice the diagnostic methods.
In addition, probes or primers that bind specifically to the RNA
component of a telomerase (or either strand of the gene for the
same) can be used in diagnostic methods to detect the presence of
telomerase nucleic acid in a sample. Primers and probes are
oligonucleotides that are complementary, and so will bind, to a
target nucleic acid. Although primers and probes can differ in
sequence and length, the primary differentiating factor is one of
function: primers serve to initiate DNA synthesis, as in PCR
amplification, while probes are typically used only to bind to a
target nucleic acid. Typical lengths for a primer or probe can
range from 8 to 20 to 30 or more nucleotides. A primer or probe can
also be labeled to facilitate detection (i.e., radioactive or
fluorescent molecules are typically used for this purpose) or
purification/separation (i.e., biotin or avidin is often used for
this purpose).
An especially preferred diagnostic method of the invention involves
the detection of telomerase RNA component sequences in cell or
tissue samples taken from patients suspected to be at risk for
cancer. Such methods will typically involve binding a labelled
probe or primer to an RNA component sequence under conditions such
that only perfectly matched (complementary) sequences bind
(hybridize) to one another. Detection of labelled material bound to
RNA in the sample can correlate with the presence of telomerase
activity and the presence of cancer cells. The diagnostic methods
of the invention may be especially useful in detecting the presence
of telomerase activity in tissue biopsies and histological sections
in which the method is carried out in situ, typically after
amplification of telomerase RNA component using specific PCR
primers of the invention.
Depending on the length and intended function of the primer, probe,
or other nucleic acid comprising sequences from the RNA component
of a telomerase, expression plasmids of the invention may be
useful. For instance, recombinant production of the full-length RNA
component of human telomerase can be carried out using a
recombinant DNA expression plasmid of the invention that comprises
a nucleic acid comprising the nucleotide sequence of the RNA
component positioned for transcription under the control of a
suitable promoter. Host cells for such plasmids can be either
prokaryotic or eukaryotic, and the promoter, as well as the other
regulatory elements and selectable markers chosen for incorporation
into the expression plasmid will depend upon the host cell used for
production.
The intact RNA components gene, i.e., the promoter, which includes
any regulatory sequences in the 5'-region of the gene, and RNA
component coding region, can be used to express the RNA component
in human cells, including human cells that have been immortalized
by viral transformation or cancer. The promoter of the RNA
component gene may be regulated, however, and for this and other
reasons, one may want to express the RNA component under the
control of a different promoter. On the other hand, the promoter of
the RNA component gene can be used independently of the RNA
component coding sequence to express other coding sequences of
interest. For instance, one could study the transcriptional
regulation of the RNA component gene by fusing the promoter of the
RNA component gene to a coding sequence for a "reporter" coding
sequence, such as the coding sequence for beta-galactosidase or
another enzyme or protein the expression of which can be readily
monitored. Thus, the promoter and other regulatory elements of the
gene for the RNA component of human telomerase can be used not only
to express the RNA component but also protein components of human
telomerase, antisense or other oligonucleotides, as well as other
gene products of interest in human cells. Expression plasmids
comprising the intact gene for the RNA component of human
telomerase can be especially useful for a variety of purposes,
including gene therapy. Those of skill in the art recognize that a
wide variety of expression plasmids can be used to produce useful
nucleic acids of the invention and that the term "plasmid", as used
herein, refers to any type of nucleic acid (from a phage, virus,
chromosome, etc.) that can be used to carry specific genetic
information into a host cell and maintain that information for a
period of time.
As indicated by the foregoing description, access to purified
nucleic acids comprising the sequence of the RNA component of a
telomerase provides valuable diagnostic and therapeutic methods and
reagents, as well as other important benefits. One important
benefit of the present invention is that the methods and reagents
of the invention can be used to isolate the RNA component and genes
for the RNA component of telomerase from any other mammalian
species that has an RNA component substantially homologous to the
human, mouse, rat, Chinese hamster or bovine coding region of the
RNA component of the present invention. The phrase "substantially
homologous" refers to that degree of homology required for specific
hybridization of an oligonucleotide or nucleic acid sequence of the
human, mouse, rat, Chinese hamster or bovine RNA component to a
nucleic acid sequence of an RNA component sequence of another
mammalian species. Given such substantial homology, those of
ordinary skill in the art can use the nucleic acids and
oligonucleotide primers and probes of the invention to identify and
isolate substantially homologous sequences.
For instance, one can probe a genomic or cDNA library to detect
homologous sequences. One can also use primers corresponding to
regions of the RNA component sequence and PCR amplification under
low or moderate stringency conditions to amplify a specific
homologous nucleic acid sequence from preparations of RNA or DNA
from a mammalian species. By using these and other similar
techniques, those of ordinary skill can readily isolate not only
variant RNA component nucleic acid from human or mouse cells but
also homologous RNA component nucleic acids from other mammalian
cells, such as cells from primates, from mammals of veterinary
interest, i.e., sheep, horses, dogs, and cats, and from other
rodents. An example of this application, wherein mouse RNA
component is isolated and sequenced, has already been described.
Example 15 also illustrates how such methodology has been used to
identify and isolate the sequence of the rat, Chinese hamster and
bovine RNA components and RNA component sequences of primates.
Further, probes or primers that comprise nucleotide sequences
encoding conserved ribonucleotides between the human, mouse, rat,
Chinese hamster, and bovine telomerase RNA components (see FIGS.
7A-7B) are especially useful for this purpose because they are most
likely to be conserved and constitute part of the RNA component in
other mammals.
The reagents of the present invention also allow the cloning and
isolation of nucleic acids encoding the protein components of human
as well as other mammalian telomerase enzymes, which have not
previously been available. Access to such nucleic acids provide
complementary benefits to those provided by the nucleic acids
comprising nucleic acid sequences of the RNA component of human or
other mammalian telomerases. For instance, and as noted above, the
therapeutic benefits of the present invention can be enhanced, in
some instances, by use of purified preparations of the protein
components of human telomerase and by access to nucleic acids
encoding the same. The nucleic acids of the invention that encode
the RNA component of human telomerase can be used to isolate the
nucleic acid encoding the protein components of human telomerase,
allowing access to such benefits. Thus, the invention provides
methods for isolating and purifying the protein components of human
telomerase, as well as for identifying and isolating nucleic acids
encoding the protein components of human telomerase. In related
aspects, the present invention provides purified human telomerase,
purified nucleic acids that encode the protein components of human
telomerase, and recombinant expression plasmids for the protein
components of human telomerase. The invention also provides
pharmaceutical compositions comprising as an active ingredient
either the protein components of human telomerase or a nucleic acid
that either encodes those protein components or interacts with
nucleic acids that encode those protein components, such as
antisense oligonucleotides, triple helix-forming oligonucleotides,
ribozymes, or recombinant DNA expression plasmids for any of the
foregoing.
The cloned RNA component of human or other mammalian telomerases
can be used to identify and clone nucleic acids encoding the
protein components of the ribonucleoprotein telomerase enzyme.
Several different methods can be employed to achieve identification
and cloning of the protein components. For instance, one can use
affinity capture of the enzyme or partially denatured enzyme using
as a affinity ligand either (1) nucleotide sequences complementary
to the RNA component to bind to the RNA component of the intact
enzyme; or (2) the RNA component to bind the protein components of
a partially or fully denatured enzyme. The ligand can be affixed to
a solid support or chemically modified (e.g., biotinylated) for
subsequent immobilization on the support. Exposure of cell extracts
containing human telomerase, followed by washing and elution of the
telomerase enzyme bound to the support, provides a highly purified
preparation of the telomerase enzyme. The protein components can
then be optionally purified further or directly analyzed by protein
sequencing. The protein sequence determined can be used to prepare
primers and probes for cloning the cDNA or identifying a clone in a
genomic bank comprising nucleic acids that encode a protein
component of telomerase.
Affinity capture of telomerase utilizing an engineered RNA
component can also be conducted using in vitro transcribed
telomerase RNA and a system for the reconstitution of telomerase
enzyme activity. See Autexier, C. and C. W. Greider (1994) Genes
& Development 8:563-575, incorporated herein by reference. The
RNA is engineered to contain a tag, similar to epitope tagging of
proteins. The tag can be an RNA sequence to which a tightly binding
ligand is available, e.g., an RNA sequence-specific antibody, a
sequence-specific nucleic acid binding protein, or an organic dye
that binds tightly to a specific RNA sequence. The tolerance of
telomerase for the tag sequence and position can be tested using
standard methods. Synthesis of the altered RNA component and the
reconstitution step of this method can also be carried on in vivo.
Affinity capture using the immobilized ligand for the RNA tag can
then be used to isolate the enzyme.
Expression screening can also be used to isolate the protein
components of the telomerase enzyme. In this method, cDNA
expression libraries can be screened with labeled telomerase RNA,
and cDNAs encoding proteins that bind specifically to telomerase
RNA can be identified. A molecular genetic approach using
translational inhibition can also be used to isolate nucleic acids
encoding the protein components of the telomerase enzyme. In this
method, telomerase RNA sequences will be fused upstream of a
selectable marker. When expressed in a suitable system, the
selectable marker will be functional. When cDNA encoding a
telomerase RNA binding protein is expressed, the protein will bind
to its recognition sequence thereby blocking translation of the
selectable marker, thus allowing for identification of the clone
encoding the protein. In other embodiments of this method, the
blocked translation of the selectable marker will allow transformed
cells to grow. Other systems that can be employed include the
"interaction trap system" described in PCT patent publication No.
WO 94/10300; the "one-hybrid" system described in Li and Herskowitz
(1993) Science 262:1870-1874, and Zervos, et al. (1993) Cell
72:223-232; and the "two-hybrid" system commercially available from
Clontech.
Telomerase RNA binding or telomerase activity assays for detection
of specific binding proteins and activity can be used to facilitate
the purification of the telomerase enzyme and the identification of
nucleic acids that encode the protein components of the enzyme. For
example, nucleic acids comprising RNA component sequences can be
used as affinity reagents to isolate, identify, and purify
peptides, proteins or other compounds that bind specifically to a
sequence contained within the RNA component, such as the protein
component of human telomerase. Several different formats are
available, including gel shift, filter binding, footprinting,
Northwestern (RNA probe of protein blot), and photocrosslinking, to
detect such binding and isolate the components that bind
specifically to the RNA component. These assays can be used to
identify binding proteins, to track purification of binding
proteins, to characterize the RNA binding sites, to determine the
molecular size of binding proteins, to label proteins for
preparative isolation, and for subsequent immunization of animals
for antibody generation to obtain antibodies for use in isolating
the protein or identifying a nucleic acid encoding the protein in a
coupled transcription/translation system.
The mouse, rat, or Chinese hamster RNA components have many
additional uses. Like the human RNA component, they can be used to
prepare transgenic animals of great value for screening and testing
of pharmaceuticals that regulate telomerase activity.
For instance, by using a plasmid, one can "knock out" the RNA
component gene or replace the natural RNA component gene with a
recombinant inducible gene in a Mus muculus embryonic stem cell and
then generate a transgenic mouse that will be useful as a model or
test system for the study of cancer, or age- or senescence-related
disease. The generation of mice and mouse cells lacking telomerase
makes it possible to determine the effects of telomerase inhibitors
directly. In addition, the toxicity of telomerase inhibitors can be
assessed in an animal model where the specific target has been
removed. Thus, any side effects of such inhibitors will be revealed
in the mouse model.
Initially a knockout mouse is generated with one of the two
telomerase alleles deleted. As an example of a knockout plasmid
that could be used for this task, a plasmid vector has been
constructed (FIG. 6) using standard cloning procedures. Sambrook,
et al. (1989) supra. In this plasmid, the entire telomerase RNA
gene has been eliminated and replaced by the bacterial neomycin
gene (neo). The restriction map of the mouse telomerase RNA genomic
region is shown in FIG. 6, as well as the sections that were cloned
into the knockout vector. This plasmid can be used to generate
knockout mice from a number of Mus musculus ES cell strains.
Mouse model systems can also be used to study telomerase regulation
and telomere length in mammals. Similar to human cells, mouse
fibroblasts show no telomerase activity during growth while
telomere length decreases until the culture passes through crisis
when telomerase activity is detected as the telomere length
stabilizes. Furthermore, telomere lengths from tissues of
individual adult mice differ between tissues. For example, testes
telomere length is significantly longer than in other tissues,
suggesting that a developmentally-regulated telomere length
increase may occur after birth. Thus the mouse offers an excellent
system in which to directly test the role of telomerase in aging
and immortalization because the effects of altered telomerase on
both cell viability and organismal development can be determined in
vivo.
The same or another mammalian system can be used to test the
effects of therapeutic and pharmaceutical compounds on telomerase
activity. Anti-telomerase compositions, including antibodies,
directed towards telomerase activity in tumors can be screened for
efficacies and side effects.
As will be apparent to those of skill in the art upon reading of
this disclosure, the present invention provides valuable reagents
relating to human or mammalian telomerases, as well as a variety of
useful therapeutic and diagnostic methods, and model systems. The
above description of necessity provides a limited sample of such
methods, which should not be construed as limiting the scope of the
invention. Other features and advantages of the invention will be
apparent from the following examples and claims.
The following examples describe specific aspects of the invention
to illustrate the invention and provide a description of the
methods used to isolate and identify the RNA component of human,
mouse, rat, Chinese hamster and bovine telomerase for those of
skill in the art. The examples should not be construed as limiting
the invention, as the examples merely provide specific methodology
useful in understanding and practice of the invention.
EXAMPLE 1
Preparation of PCT-amplifiable cDNA
RNA was obtained from 293 cells by guanidine-thiocyanate extraction
of from purified telomerase fractions by phenol/chloroform
extractions. The total RNA from 293 cells was size fractionated on
a 2% agarose gel, and the RNA below 500 bp was isolated.
First strand cDNA synthesis was performed with Superscript.TM. II
reverse transcriptase obtained from Bethesda Research Laboratories
(BRL). About 0.5 to 1.0 .mu.g RNA was mixed with about 40 ng of
random primer (6 mer) in water at a total volume of 11 .mu.l. The
solution was heated for 10 min. at 95.degree. C. and then cooled on
ice for 5-10 min. The denatured nucleic acid was collected by
centrifugation. The denatured RNA and primer mixture were then
resuspended by adding, in the order shown: 4 .mu.l 5.times.1st
strand synthesis buffer; 2 .mu.l 0.1M dithiothreitol (DTT); 1 .mu.l
RNAsin (Pharmacia); and 1 .mu.l dNTP (0.125 mM each for 0.5 mM
total concentration). The reaction mixture was incubated at
42.degree. C. for 1 min., and then, 1 .mu.l (200 units) of
Superscript.TM. II RTase (BRL) was added and mixed into the
reaction, which was then incubated for 60 min. at 42.degree. C. The
resulting reaction mixture, containing the newly synthesized cDNA
was placed on ice until second strand synthesis was performed.
Second strand cDNA synthesis was performed as follows: About 20
.mu.l of the reaction mixture from the first strand cDNA synthesis
reaction mixture (from above) was mixed with, in the order shown,
the following components: 111.1 .mu.l of water; 16 .mu.l of
10.times. E. coli DNA ligase buffer; 3 .mu.l of dNTP (2.5 mM each
stock); 1.5 .mu.l of E. coli DNA ligase (15 units from BRL); 7.7
.mu.l of E. coli DNA polymerase (40 units from Pharmacia); and 0.7
.mu.l of E. coli RNase H (BRL). The resulting solution was gently
mixed and incubated for 2 hours at 16.degree. C., at which time 1
.mu.l (10 units) of T4 DNA polymerase was added to the reaction
tube and incubation continued for 5 min. at the same temperature
(16.degree. C. ). The reaction was stopped, and the nucleic acid
was collected by extracting the reaction with phenol/chloroform
twice, precipitating the nucleic acid with ethanol, and
centrifuging the reaction mixture to pellet the nucleic acid.
The cDNA pellet collected by centrifugation was resuspended in 20
.mu.l of TE buffer and ligated to a double-stranded oligonucleotide
called "NotAB" composed of two oligonucleotides (NH2 is an amino
blocking group):
NotA: 5'-pATAGCGGCCGCAAGAATTCA-NH2 (SEQ ID NO:14)
NotB: 5'-TGAATTCTTGCGGCCGCTAT-3' (SEQ ID NO:15)
The double-stranded oligonucleotide was made by mixing 50.mu. of
NotA oligonucleotide (100 pmol) with 50 .mu.l of NotB
oligonucleotide (100 pmol) in 46.25 .mu.l of water, heating the
resulting solution for 5 min. at 95.degree. C., and adding 3.75
.mu.l of 20.times. SSC buffer while the solution was still hot. The
tube containing the mixture was then placed in a beaker containing
hot water (at a temperature of about 70 to 75.degree. C.), the
temperature of which was allowed to drop slowly to below 15.degree.
C., so that the two oligonucleotides could hybridize to form the
double-stranded oligonucleotide NotAB. The resulting nucleic acid
was collected by precipitation and centrifugation.
The double-stranded NotAB oligonucleotide was resuspended in about
30 .mu.l TE buffer and then ligated to the cDNA in a reaction
mixture containing 10 .mu.l of the cDNA preparation described
above, about 50 pmol (calculated by OD260) of NotAB; 2 .mu.l of
10.times. T4 DNA ligase buffer; 1.2 .mu.l of T4 DNA ligase; 0.2
.mu.l of 10 mM ATP; and water in a total volume of 20 .degree.l by
incubating the reaction mixture at 16.degree. C. overnight. The
reaction was then heat-inactivated by heating the reaction mixture
for 10 min. at 65.degree. C. About 1 to 2 .mu.l of the resulting
mixture was typically used for PCR amplification; one can amplify
the ligation mixture for 10 to 15 cycles (94.degree. C., 45
seconds; 60.degree. C., 45 seconds; and 72.degree. C., 1.5 min.)
and save as a stock, as described in Example 2.
EXAMPLE 2
PCR amplification of cDNA
The cDNA was routinely amplified by preparing an amplification
reaction mixture composed of 5 .mu.l of 10.times. PCR buffer (500
mM KCL; 100 mM Tris, ph=8.3; and 20 mM MgCl.sub.2 ; 5-8 .mu.l of
dNTP (2.5 mM each); 1 .mu.l of Taq polymerase
(Boehringer-Mannheim); 0.1 .mu.l of gene 32 protein
(Boehringer-Mannheim); 6 .mu.l of Not B primer (20 .mu.M stock); 2
.mu.l of the cDNA (prepared as described in Example 1), and water
to 50 .mu.l. This mixture was then overlaid with 50 to 100 .mu.l of
mineral oil, and PCR amplification was performed for 10 to 15
cycles of 94.degree. C., 45 seconds; 60.degree. C., 45 seconds; and
72.degree. C., 1.5 min. After amplification, the reaction mixture
was extracted with phenol/chloroform, and the amplified nucleic
acid was precipitated with ethanol and collected by centrifugation.
The precipitate was then dissolved in 100 .mu.l of TE buffer to
prepare a stock solution.
EXAMPLE 3
PCR amplification for cyclic selection
To make PCR product for cyclic selection, about 1 .mu.l of a stock
solution prepared as described in Example 2 was amplified in 50
.mu.l of PCR reaction mixture prepared as described in Example 2,
except that 21-24 cycles of primer annealing, extension, and
denaturation of product were conducted. After amplification,
reaction mixtures were extracted with phenol/chloroform,
precipitated with ethanol, and collected by centrifugation. Product
yield was estimated by staining with ethidium bromide after agarose
gel electrophoresis of a small aliquot of the reaction mixture.
Typically, about 2 .mu.g of the nucleic acid product were used for
cyclic selection.
After cyclic selection, described in Example 4, about 1 to 2 .mu.l
of the selected "pull-down" products (out of a total volume of 20
.mu.l) were PCR amplified as described in Example 2 for 22 cycles,
precipitated with ethanol, and collected by centrifugation in
preparation for further cyclic selection.
EXAMPLE 4
Positive selection of PCT-amplified cDNA
For the positive selection step of the cyclic selection process
used to clone the RNA component of human telomerase, about 2 .mu.g
of the PCR-amplified cDNA were diluted into 25 .mu.l of TE buffer
and then mixed with 1.25 .mu.l of 20.times. SSC and the resulting
solution heated to 95.degree. C. for 3 min. The temperature was
lowered to 60.degree. C. for 5 min., and one .mu.l (0.1
.mu.g/.mu.l) of the R2 or R4 biotinylated probe was added. The
sequences of these probes are shown below. The probes are
O-methyl-RNA probes, so U is O-methyl-uridine, A is
O-methyl-riboadenine, G is O-methyl-riboguanine, and I is
inosine.
R2: 5'-UUAGGGUUAGII-biotin
R4: 5'-AUUGGGUUAUII-biotin
The R2 probe is specific for the telomere repeat, and the R4 probe
is specific for RNase P, which was used to track the effectiveness
and efficiency of the cyclic selection process. By carrying out a
cyclic selection simultaneously but separately for RNase P RNA, a
molecule of known sequence, one can have greater confidence that
the cyclic selection process is functioning properly with respect
to the molecule of interest, in this case the RNA component of
human telomerase.
After either the R2 or R4 probe was added to the mixture at
65.degree. C., the temperature of the hybridization reaction
mixture was lowered to 30.degree. C. by incubating the mixture at
that temperature for 5 min., and then the reaction mixtures were
further lowered to a temperature of 14.degree. C. by incubating at
that temperature for 60 min. Finally, the mixture was incubated at
4.degree. C. for 2-12 hours.
The entire hybridization reaction mixture for each sample (R2 or
R4) was added to 400 .mu.l of 0.5.times. SSC at 4.degree. C. and
then added to a tube of ice-cold magnetic beads, which were
purchased from Promega and pre-washed four times with 0.5.times.
SSC before use. The resulting mixture was incubated 30 min. at
4.degree. C. to ensure complete binding to the magnetic beads. Each
reaction tube was then incubated briefly at room temperature on the
magnetic stand (Promega) to pull down the beads. The beads were
resuspended in cold 0.5.times. SSC (600 .mu.l) and placed (in a
tube) on ice. The samples were washed three more times with
0.5.times. SSC in this manner. Nucleic acid was eluted from the
beads by resuspending the beads in 100 .mu.l of water and
incubating for 2 min. at 65.degree. C. before placing the beads
back on the magnetic stand for collection. This process was
repeated three more times; the last time, the resuspended beads
were incubated for 5 min. at 65.degree. C. before placing the beads
on the magnetic stand for collection. All of the 100 .mu.l
supernatants (for each sample) were pooled and dried down to 20
.mu.l in a SpeedVac.TM. centrifuge. The recovered DNA was then PCR
amplified for another round of amplification and selection. After
each amplification, the PCR products were phenol-chloroform
extracted twice, ethanol precipitated, and resuspended in 20 .mu.l
of TE buffer.
Typically, PCR amplifications were verified by agarose gel
electrophoresis. In addition, a variety of controls were used to
monitor the cyclic selection process. As one control, PCR "arms"
(oligonucleotides of defined sequence that serve as primer
hybridization sites) were placed on a nucleic acid that comprised a
neomycin resistance-conferring gene. The resulting nucleic acid was
mixed with the PCR-amplified cDNA and monitored at each selection
by quantitative PCR. As another control, RNase P was followed in
both the RNase P selected and the telomerase RNA component selected
libraries.
EXAMPLE 5
RT-PCR protocol
The first strand cDNA was made in substantial accordance with the
procedure described in Example 1. Basically, RNA was purified from
each telomerase fraction containing 0.1 to 1 .mu.g RNA; typical,
about one-third to one-fifth of the RNA made from a 300 .mu.l
fraction was used. The RNA was mixed with 40 to 80 ng random
hexamer in 10 .mu.l, denatured for 10 min. at 95.degree. C. (using
a thermal-cycling instrument), and chilled on ice. The denatured
RNA and 6-mer were added to a reaction mixture containing 4 .mu.l
of 5.times.1st strand synthesis buffer supplied by the manufacturer
of the reverse transcriptase (RTase, purchased from BRL), 2 .mu.l
of 0.1 DTT, 1 .mu.l of 10 mM dNTP (each), 1 .mu.l of RNase
inhibitor (Pharmacia), and water to a total volume of 9 .mu.l. The
combined mixture was placed into a 42.degree. C. water bath. After
1-2 min. incubation, 1 .mu.l of Superscript.TM. II RTase (BRL) was
added to the mixture. The incubation was continued for 60 min. at
42.degree. C. The reaction was stopped by heating the tube for 10
min. at 95.degree.-98.degree. C. The first strand cDNA was
collected by brief centrifugation, aliquoted to new tubes, quickly
frozen on dry ice, and stored at -80.degree. C. or used
immediately.
Example 6
PCR amplification of cDNA with a specific primer set
For a 20 .mu.l PCR reaction with radioactively labeled nucleotides,
1 .mu.l of the cDNA prepared in accordance with the procedure of
Example 5 was mixed with 20 pmol of primer 1, 20 pmol of primer 2,
2.5 .mu.l of 2.5 mM dNTP, 5 .mu.Ci of alpha-.sup.32 p-dATP, 2 units
of Taq polymerase (Boehringer-Mannheim), 0.2 .mu.g of T4 gene 32
protein (Boehringer-Mannheim), 2 .mu.l of 10.times. buffer (500 mM
KCL, 100 mM Tris-HCl-pH8.3, and 20 mM MgCl.sub.2), and water to a
total volume of 20 .mu.l. One drop of mineral oil was then added to
the tube.
The PCR amplification conditions for the telomerase RNA component
clone were: 94.degree. C. for 45 sec., 60.degree. C. for 45 sec.,
72.degree. C. for 1.5 min. The number of cycles differed depending
on the type of purified materials used for RNA preparation but
typically range from 18 to 25 cycles. As for all quantitative
RT-PCR, several reactions with differing cycles were run for each
sample to determine when the PCR amplification became saturated and
non-linear.
For the RNase P used as a control, the PCR amplification conditions
were: 94.degree. C. for 45 sec., 50.degree. C. for 45 sec., and
72.degree. C. for 1.5 min. Again, the number of cycles ranged from
15 to 22 cycles, depending on the nature of the samples. The
sequences of the primers used for RNase P amplification are shown
below:
P3: 5'-GGAAGGTCTGAGACTAG-3' (SEQ ID NO:16)
P4: 5'-ATCTCCTGCCCAGTCTG-3' (SEQ ID NO:17)
The PCR product obtained with these two primers is about 110 bp in
size.
After PCR, the products (5 to 10 .mu.l of the reaction mixture)
were loaded onto a 6% native polyacrylamide gel and
electrophoresed. After electrophoresis, the gel was dried and
exposed to a PhosphorImager.TM. cassette or to autoradiographic
film for analysis.
EXAMPLE 7
Cloning the gene for the RNA component of human telomerase
The procedures used to clone the gene for the RNA component of
human telomerase were carried out as generally described in
Maniatis, et al., Laboratory Molecular Cloning Manual. A genomic
DNA library of DNA from the human lung fibroblast cell line WI-38
inserted into phage lambda vector FIXII was purchased from
Stratagene. The phage were plated at a concentration of about
25,000 plaques per plate onto three sets of 15 (150 mm) plates. The
plates were made with NZY agar and NZY top agarose; the cells used
for the phage transformation were XL1BlueMRAP2 cells; and the
transformants were grown overnight for about 16 hours at 37.degree.
C. The plates were then chilled at 4.degree. C. for about an hour,
and then the plaques were "lifted" onto C/P nylon circles (filter
paper from Bio Rad). This process was repeated to produce a
duplicate set of lifted filters. The filters (in duplicate) were
denatured, neutralized, equilibrated in 6.times. SSC buffer,
exposed to UV irradiation to cross-link the nucleic acid to the
filter, and then dried on blotter paper.
Prehybridization was conducted for one hour at 37.degree. C. in 50%
formamide buffer. The filters were probed with an .about.218 bp,
radioactively-labeled, NotI fragment from clone pGRN7, which has
been isolated by electroelution from a 5% polyacrylamide gel after
separation by electrophoresis and then nick-translated with
alpha-.sup.32 p-dCTP using a nick-translation kit from
Boehringer-Mannheim Biochemicals in accordance with the
manufacturer's instructions. About 25 ng (.about.10 .mu.Ci label)
of the probe were used per filter, and hybridization was conducted
overnight at 37.degree. C. in 50% formamide hybridization buffer.
After hybridization, the filters were washed at room temperature
six times; the first three washes were with 6.times. SSC containing
0.1% SDS, and the last three washes were with 6.times. SSC alone.
After an initial exposure of several duplicate filters in a
PhosphorImager.TM. cassette to check hybridization efficiency and
signal strength, the filters were washed at 65.degree. C. in
0.5.times. SSC. The filters were then placed under Kodak XAR5 film
using two intensifier screens and then allowed to expose the film
for about 100 hours at -70.degree. C.
One strong signal emanated from the filter containing a phage,
later designated 28-1, comprising the gene for the RNA component of
human telomerase. The plaque corresponding to the signal observed
on the filter was used to make secondary plates, so that an
isolated plaque (confirmed by probing with labeled pGRN7 nucleic
acid) could be cultured for large-scale isolation of the phage DNA.
Phage 28-1, available from the American Type Culture Collection
under ATCC accession No. 75925, comprises an .about.15 kb insert
and comprises several restriction fragments that contain sequences
that hybridize with RNA component sequences on pGRN7: a 4.2 kb
EcoRI restriction enzyme fragment; a 4.5 kb ClaI restriction enzyme
fragment, and a 2.5 kb HindIII-SacI restriction enzyme fragment.
The latter fragment comprises the entire .about.560 nucleotide
sequence of the RNA component shown above and is believed to
comprise the complete gene for the RNA component. The plasmid
comprising the 2.5 kb HindIII-SacI restriction enzyme fragment in
the pBluescript vector was designated plasmid pGRN33 and is
available from the American Type Culture Collection under ATCC
accession No. 75926. To the extent the human gene may comprise
sequences other than those on the 2.5 kb fragment, those sequences
can be isolated from phage 28-1 or from other phage clones
identified by probing with the 2.5 kb fragment (or another probe of
the invention).
The restriction enzyme fragments noted above were prepared in
separate restriction enzyme digests; the products of the digests
were separated by electrophoresis on a 0.7% agarose gel or, for the
.about.2.5 kb fragment only, a 3% polyacrylamide gel; and the
desired bands were cut from the gel and prepared for subcloning
either by using the GeneClean.TM. Kit II (from Bio101, Inc.) or by
electroelution into Spectropor #2 dialysis tubing in 0.1.times. TBE
at 100 V for two hours (for the .about.2.5 kb fragment only).
These restriction enzyme fragments were subcloned into E. coli
expression/mutagenesis plasmids derived from pUC-based plasmids or
from pBluescriptII plasmids that also comprise an SV40 origin of
replication (but no SV40 promoter activity). The resulting plasmids
can be used to prepare altered (mutated) RNA component nucleic
acids for introduction into human or other eukaryotic cells for a
variety of purposes, as described above in the Description of the
Preferred Embodiments.
EXAMPLE 8
Antisense plasmids for the RNA component of human telomerase
Antisense expression plasmids were prepared by PCR amplification of
RNA component cDNA using the following primer sets: (1) NotB and
G1, which produces an antisense nucleic acid that is smaller than
the cDNA insert in the plasmid; and (2) NotB and R3C, which
produces a full-length (relative to the insert in the plasmid)
antisense nucleic acid. The nucleotide sequence of NotB is shown in
Example 1, above: the nucleotide sequences of the G1 and R3 primers
are shown below.
G1: 5'-GAGAAAAACAGCGCGCGGGGAGCAAAAGCA-3' (SEQ ID NO:18)
R3C: 5'-GTTTGCTCTAGAATGAACGGTGGAAG-3' (SEQ ID NO:19)
After PCR amplification, the amplified fragments were cloned into
an .about.10 kb expression plasmid at a PmlI site: the plasmid
comprises puromycin resistance-conferring, DHFR, and hygromycin B
resistance-conferring genes as selectable markers, the SV40 origin
of replication; the inducible human metallothionein gene promoter
positioned for expression of the antisense strand of the gene for
the RNA component of human telomerase (one could also use a
stronger promoter to get higher expression levels), and the SV40
late poly A addition site.
The resulting plasmids (designated pGRN42 for the NotB/G1 product
and pGRN45 for the NotB/R3C product) were transfected by the
calcium phosphate procedure (see Maniatis, et al., supra) into the
fibrosarcoma cell line HT1080. HT1080 cells are normally immortal;
expression of the antisense RNA for the RNA component of human
telomerase should prevent the RNA component of human telomerase
from association with the protein components, blocking the
formation of active telomerase and rendering the cells mortal.
EXAMPLE 9
Mouse Telomerase Activity Purification
Forty liters of mouse FM3A cells were grown in suspension in DMEM
medium supplemented with 10% calf serum to a density of 0.5
10.sup.5 cells/ml. S-100 cytoplasmic extracts were prepared as
described (Counter, et al. (1992) EMBO J. 11:1921-1929; Prowse, et
al. (1993) supra) and glycerol and NaCl were added to a final
concentration of 10% and 0.1M, respectively. For purification of
mouse telomerase activity, the S-100 extracts were passed through
30 ml of a DEAE-agarose column previously equilibrated in 1.times.
hypo-buffer (Prowse, et al. (1993) supra) containing 0.1M NaCl as
well as several protease inhibitors. After collection, the flow
through the column was washed with 3 volumes of hypo-buffer-0.1M
NaCl. Hypo-buffer containing increasing NaCl concentrations (0.2M,
0.35M, 0.5M and 0.7M) was used as eluant, and 100 ml fractions were
collected at each salt concentration. Telomerase activity eluted at
0.35M NaCl. This telomerase-containing fraction was then diluted
with 1.times. hypo buffer to 0.1M NaCl final concentration and
loaded on a 10 ml of a spermine-agarose column previously
equilibrated with 1.times. hypo-buffer-0.1M NaCl. After collecting
the flow through, the wash and elution steps were carried out as
described previously (Prowse, et al. (1993) supra; the volume of
each eluate was 20 ml and activity eluted between 0.35M and 0.5M
NaCl. The lower salt fraction, containing most of the telomerase
activity was loaded onto an octyl-sepharose column equilibrated
with 1.times. hypo-buffer-0.1M NaCl. The column was washed as
described previously and eluted with 1.times. hypo-buffer
containing 1% triton X-100, fractions of 3 ml were collected, most
of the telomerase activity was in fraction number 3.
Twenty .mu.l of each fraction were used to assay telomerase
activity as described (Prowse, et al. (1993) supra). RNA was
prepared from all the column fractions by extracting 100 .mu.l of
each fraction first with one volume of phenol and then with one
volume of phenol/chloroform followed by ethanol precipitation
(Sambrook, et al., (1989) Molecular Cloning--a laboratory manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). RNA was
run on a 60% acrylamide/urea gel at 20 W. RNA was electroblotted to
a Hybond N+ membrane and hybridized with a 300 bp PCR fragment of
mouse telomerase RNA (mTR) gene. Hybridization was done at
65.degree. C. in 1% BSA, 200 mM NaPO.sub.4, 15% formamide, 1 mM
EDTA and 7% SDS. Filters were washed in 0.4.times. SSC, 0.1% SDS at
65.degree. C.
EXAMPLE 10
Mouse Genomic Library Screening
A genomic library from D3 embryonic stem cells cloned in .lambda.
EMBL3 was used for screening. Three to four genomes were plated for
the primary screening using the strain LE392 (P2) as plating cells.
A PCR product of 450 bp of the human telomerase RNA (hTR) gene was
gel purified and radiolabeled by hexameter labeling (Sambrook, et
al. (1989) supra) using high specific activity (3000 Ci/mmol)
dNTPs. The sequences of the primers used to generate the probe were
as follows:
u3b: 5'-GCCTGGAGGGGTGGTGGCCATTTTTTG-3' (SEQ ID NO:20)
ol3: 5'-GATCGGCGTTCCCCCCACCAAC-3' (SEQ ID NO:21).
The mouse library was probed with the human sequence at 55.degree.
C. in a moderate stringency buffer (Sambrook, et al. (1989) supra
containing 1% BSA, 500 mM NaPO.sub.4, 15% formamide, 1 mM EDTA and
7% SDS. The filters were washed at 55.degree. C. in 0.4.times. SSC
and 0.1% SDS. Positive plaques were re-plated and probed again to
further purify the positive lambda clones. After the isolation of
single positive phage clones, 50 ml LE392 (P2) culture was infected
and DNA was prepared using the Qiagen Kit for lambda DNA
purification. A restriction map with four different enzymes that do
not cut lambda DNA sequences was done for each positive clone. Four
different clones had the same pattern with each enzyme, moreover,
the same size restriction fragment for each enzyme hybridized to
the human probe in a Southern blot. A 5 Kb EcoRI genomic fragment
containing the homology region of the human RNA was cloned into
KS.sup.+ Bluescript vector and a further restriction map was done.
The restriction enzyme PstI cut within the homology region. Both
PstI fragments were cloned and sequenced.
EXAMPLE 11
RT-PCR Protocol
A 3 .mu.g sample of total RNA from the FM3A cell line was mixed
with 8 ng of random hexamers in a final volume of 10 .mu.l,
denatured at 95.degree. C. for 10 minutes and chilled on ice. The
annealing mixture was added to the reverse transcription mixture
under the same conditions as for primer extension. Reverse
transcriptase was not added to a control tube. Incubation was for
60 minutes at 50.degree. C. The reactions were stopped by heating
the tubes at 95.degree. C. for 10 minutes. A 1 .mu.l aliquot of
each first strand cDNA was used for each PCR reaction. PCR
reactions contained 1.times. PCR buffer provided with Taq
polymerase, 5 mM dNTPs, 100 ng of each primer, 0.1 .mu.g of T4 gene
32 protein and 2 units of Taq polymerase from Perkin-Elmer. The
conditions of PCR amplification were: 94.degree. C. for 1 minute;
60.degree. C. for 45 seconds; and 72.degree. C. for 1.5 minutes.
Typically, 35 to 40 cycles were carried out for a first
amplification. For nested amplification, 1 .mu.l of the first PCR
was used in a second PCR. For cloning, the PCR products were phenol
extracted, precipitated and digested with restriction enzymes NotI
and SalI, and cloned in KS+ Bluescript that was previously digested
with NotI and SalI. Primers used were: First amplification:
mTR5b: 5'-CGTCGACTAGGGTCGAGGGCGGCTAGGCCT-3' (SEQ ID NO:22)
mTR3: 5'-GGAGGCGGCCGCAGACGTTTGATTTTTTGAGGC-3' (SEQ ID NO:23)
Second amplification
mTR5b: (see above)
nest B: 5'-GGAGGCGGCCGCAGACGTTTGTTTTTTGAGGC-3' (SEQ ID NO:24).
EXAMPLE 12
Mouse Inhibition/Elongation Experiments
Each oligonucleotide used in these experiments-was gel purified on
a 10% acrylamide-urea gel; the band corresponding to the unit
length size was excised and eluted from the gel in water. After
elution, oligonucleotides were further purified using a NAP-5
column (Pharmacia, Inc.). The concentration of each oligonucleotide
was determined by measuring the O.D. at 260 nm. For both inhibition
and priming experiments, the indicated amount of oligonucleotide
was preincubated on ice for 30 min. with 20 .mu.l of a DEAE-agarose
fraction containing telomerase activity, either pretreated or not
with DNase-free RNase. After pre-incubation, 20 .mu.l of 2.times.
telomerase reaction mix (100 mM Tris-acetate, pH 8.5; 100 mM
potassium acetate, 4 mM dTTP, 4 mM dATP, 2 mM MgCl.sub.2, 2 mM
spermidine, 2 mM EGTA, 10 mM 2-mercaptoethanol, 20 .mu.Ci of a-32P
dGTP (800 Ci/mmol, New England Nuclear)) was added and telomerase
reactions were carried out as described. For the inhibition
studies, 1.0 .mu.g of telomeric oligonucleotide (T.sub.2
AG.sub.3).sub.3 was also added to the 2.times. reaction mix for the
inhibition studies. All ddNTPs were used at a final concentration
of 2 mM.
The sequence of the oligonucleotides used for inhibition and
elongation experiments were as follows:
MI-2: ATGAAAATCAGGGTTAGG (SEQ ID NO:25)
MP-1: CCACAGCTAATGAAAATC (SEQ ID NO:26)
MP-4: CCACAGCTAATGAAAATCAGGGTTAGG (SEQ ID NO:27)
MI-3: TCACGTTCAAGGGTTAGG (SEQ ID NO:28)
MI-5: ATGAAAATCGCTACCTAA (SEQ ID NO:29)
MP-3: CCCACAGCTAATGAAAAT (SEQ ID NO:30)
MP-4: CCCCACAGCTAATGAAAA (SEQ ID NO:31).
EXAMPLE 13
Northern Blots of Cell Culture and Tissues
Total RNA from different mouse tissues was purchased from Clontech,
and total RNA from pre-crisis and post-crisis Mus spretus
fibroblasts was prepared from 90 mm tissue culture plates as
described (Sambrook, et al., 1989). RNA concentration was
determined measuring the absorbance at 260 nm in a
spectrophotometer. Twenty .mu.g of each RNA was run on a 6%
acrylamide-7M urea gel and transferred to Hybond N+ membrane.
Hybridization with the mouse telomerase RNA (mTR) probe was done in
high stringency conditions as described in Sambrook, et al. (1989)
supra.
EXAMPLE 14
Construction of a mouse knockout plasmid
FIG. 6 shows the restriction map for approximately 15 kb
surrounding the mouse telomerase RNA component gene in the mouse
genome. For targeting a standard mouse knock out, vector pPNT was
used (plasmid structure shown at bottom of the figure). To create
the specific knockout plasmid, a 3.3 kb fragment of genomic DNA 5'
region of the mouse gene was altered to add a SacI (Sc) site by
site-directed mutagenesis. The 3.3 kb fragment was cloned into the
XbaI site downstream from the neo gene in pPNT. In addition, a 4.0
kb genomic fragment from the 3" region flanking the mouse
telomerase RNA gene was altered to add a SacI restriction site.
This fragment was then cloned into the upstream XhoI site in the
pPNT vector.
EXAMPLE 15
Identification and Isolation of RNA Component Nucleic Acids from
Other Non-human Mammals
To illustrate how the reagents of the invention can be used to
identify and isolate substantially homologous nucleic acids from
other mammalian species, PCR primers complementary to human RNA
component sequences were used to amplify homologous sequences in a
PCR. An illustrative primer pair used to demonstrate this aspect of
the invention is composed of primer +10, which has the sequence
5'-CACCGGGTTGCGGAGGGAGG-3' (SEQ ID NO:32), and primer R7 which has
the sequence 5'-GGAGGGGCGAACGGGCCAGCA-3' (SEQ ID NO:33). Genomic
DNA was prepared from chimpanzee, squirrel monkey, rhesus monkey,
and baboon tissue and dissolved in TE buffer at a concentration of
about 0.5-4 mg/ml.
For each tissue type, a PCR mixture was prepared, which mixture
comprised: 1 .mu.L of genomic DNA, 48 .mu.L of Master Mix (Master
Mix is composed of 1.times. TaqExtender.TM. buffer from Stratagene,
200 .mu.M of each dNTP, and 0.5 .mu.M of each primer), and 0.5
.mu.L of a 1:1 mixture of Taq polymerase (5 units/.mu.L,
Boehringer-Mannheim):Tth polymerase (TaqExtender.TM. polymerase,
from Stratagene). The reaction tubes were loaded onto a thermal
cycler, which was programmed to first heat the reaction mixture at
94.degree. C. for 5 minutes and then to perform 27 cycles of
incubations at 94.degree. C. for 30 sec., 63.degree. C. for 10
sec., and 72.degree. C. for 45 sec. After the amplification
reaction was complete, about 10 .mu.L of each reaction mixture were
loaded onto a 2% agarose gel for electrophoresis. After
electrophoresis, staining of the gel, and UV irradiation, one could
observe that each reaction mixture contained a band of the
predicted (.about.200 bp) size. Nucleic acids from these bands can
be cloned and sequenced and the remainder of the RNA component
genes from each of these mammalian species can be cloned as
described above for the gene for the RNA component of human
telomerase or mouse telomerase.
A similar procedure was used to clone and sequence the RNA
components of rat, Chinese hamster and bovine telomerase. PCR
primers were designed to the conserved regions between the human
and the mouse RNA components. In addition to mouse genomic
sequence, a Sal1 restriction site was added onto the 5' primer and
Not1 restriction site added onto the 3' primer to allow efficient
cloning. The sequence of the primers are:
mTR 5': 5'-CGTCGACTAGCGCTGTTTTTCTCGCTGACT-3' (SEQ ID NO:34)
Mtr 3': 5'-GGAGGCGGCCGCAGGTGCACTTCCCACAGCTCAG (SEQ ID NO:35).
These primers were used to PCR a specific sequence of the rat
telomerase RNA component. Genomic DNA was isolated using standard
procedures (Sambrook, et al., supra), and a region of the rat
telomerase RNA gene was amplified with these two primers using
procedures described in Sambrook, et al., supra. The PCR fragment
was cloned and sequenced.
The sequence obtained between the PCR primers (269 nucleotides)
showed a 74% homology to the mouse telomerase RNA gene. Since the
sequence that was amplified between the primers was not used to
select this clone, the 74% homology indicates that the correct gene
had been cloned. To clone the full length of the gene, a genomic
library was probed with the cloned rat fragment. Five positive
clones were identified when five genome equivalents were screened.
This suggests that the rat gene exists as a single copy in the rat
genome, like the mouse and human genes in their genomes. The entire
sequence of the rat gene is shown in FIGS. 7A-7B (SEQ ID NO:5).
Using the same primers described above, appropriate sized PCR
fragments have been generated using genomic DNA from Cow, Mink,
Chinese Hamster, African Green Monkey and the African horned frog
Xenopus. Thus primers to conserved sequences in the human and mouse
telomerase RNA genes can be generally useful in cloning telomerase
RNA genes from many different mammalian species and even from other
vertebrates. The entire sequences of Chinese hamster (SEQ ID NO:43)
and cow (SEQ ID NO:44) have also been cloned using a procedure
identical to the one used to clone the rat sequence (FIGS.
7A-7B).
EXAMPLE 16
Secondary structure of the RNA components
The cloned sequences for the human, mouse, rat, Chinese hamster and
bovine telomerase RNA components have been used to determine a
putative secondary structure folding of these RNA sequences (FIGS.
8A-8E). This secondary structure is important because it carries
out the function of the RNA.
The foregoing examples describe various aspects of the invention
and how certain nucleic acids of the invention were made. The
examples are not intended to provide an exhaustive description of
the many different embodiments of the invention encompassed by the
following claims.
__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 45 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 2425 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GATCAGTTAGAAAGTTACTAGTCCACATATAAAGTGCCAAGTCTTGTACTCAAGATTATA60
AGCAATAGGAATTTAAAAAAAGAAATTATGAAAACTGACAAGATTTAGTGCCTACTTAGA120
TATGAAGGGGAAAGAAGGGTTTGAGATAATGTGGGATGCTAAGAGAATGGTGGTAGTGTT180
GACATATAACTCAAAGCATTTAGCATCTACTCTATGTAAGGTACTGTGCTAAGTGCAATA240
GTGCTAAAAACAGGAGTCAGATTCTGTCCGTAAAAAACTTTACAACCTGGCAGATGCTAT300
GAAAGAAAAAGGGGATGGGAGAGAGAGAAGGAGGGAGAGAGATGGAGAGGGAGATATTTT360
ACTTTTCTTTCAGATCGAGGACCGACAGCGACAACTCCACGGAGTTTATCTAACTGAATA420
CGAGTAAAACTTTTAAGATCATCCTGTCATTTATATGTAAAACTGCACTATACTGGCCAT480
TATAAAAATTCGCGGCCGGGTGCGGTGGCTCATACCTGTAATCCCAGCACTTTGGGAGGC540
CGAAGCGGGTGGATCACTTGAGCCCTGGCGTTCGAGACCAGCCTGGGCAACATGGTGAAA600
CCCCCGTCTCTACTAAAAACACAAAAACTAGCTGGGCGTGGTGGCAGGCGCCTGTAATCC660
CAGCTACTCAGGAGGCTGAGACACGAGAATCGCTTGAACCCGGGAGCAGAGGTTGCAGTG720
AGCCGAGATCACGCCACTAGACTCCATCCAGCCTGGGCGAAAGAGCAAGACTCCGTCTCA780
AAAAAAAAAATCGTTACAATTTATGGTGGATTACTCCCCTCTTTTTACCTCATCAAGACA840
CAGCACTACTTTAAAGCAAAGTCAATGATTGAAACGCCTTTCTTTCCTAATAAAAGGGAG900
ATTCAGTCCTTAAGATTAATAATGTAGTAGTTACACTTGATTAAAGCCATCCTCTGCTCA960
AGGAGAGGCTGGAGAAGGCATTCTAAGGAGAAGGGGGCAGGGTAGGAACTCGGACGCATC1020
CCACTGAGCCGAGACAAGATTCTGCTGTAGTCAGTGCTGCCTGGGAATCTATTTTCACAA1080
AGTTCTCCAAAAAATGTGATGATCAAAACTAGGAATTAGTGTTCTGTGTCTTAGGCCCTA1140
AAATCTTCCTGTGAATTCCATTTTTAAGGTAGTCGAGGTGAACCGCGTCTGGTCTGCAGA1200
GGATAGAAAAAAGGCCCTCTGATACCTCAAGTTAGTTTCACCTTTAAAGAAGGTCGGAAG1260
TAAAGACGCAAAGCCTTTCCCGGACGTGCGGAAGGGCAACGTCCTTCCTCATGGCCGGAA1320
ATGGAACTTTAATTTCCCGTTCCCCCCAACCAGCCCGCCCGAGAGAGTGACTCTCACGAG1380
AGCCGCGAGAGTCAGCTTGGCCAATCCGTGCGGTCGGCGGCCGCTCCCTTTATAAGCCGA1440
CTCGCCCGGCAGCGCACCGGGTTGCGGAGGGAGGGTGGGCCTGGGAGGGGTGGTGGCCAT1500
TTTTTGTCTAACCCTAACTGAGAAGGGCGTAGGCGCCGTGCTTTTGCTCCCCGCGCGCTG1560
TTTTTCTCGCTGACTTTCAGCGGGCGGAAAAGCCTCGGCCTGCCGCCTTCCACCGTTCAT1620
TCTAGAGCAAACAAAAAATGTCAGCTGCTGGCCCGTTCGCCCCTCCCGGGACCTGCGGCG1680
GGTCGCTGCCCAGCCCCCGAACCCCGCCTGGAGGCCGCGGTCGGCCGGGGCTTCTCCGGA1740
GGCACCCACTGCCACCGCGAAGAGTTGGGCTCTGTCAGCCGCGGGTCTCTCGGGGGCGAG1800
GGCGAGGTTCACCGTTTCAGGCCGCAGGAAGAGGAACGGAGCGAGTCCCGCGCGCGGCGC1860
GATTCCCTGAGCTATGGGACGTGCACCCAGGACTCGGCTCACACATGCAGTTCGCTTTCC1920
TGTTGGTGGGGGGAACGCCGATCGTGCGCATCCGTCACCCCTCGCCGGCAGTGGGGGCTT1980
GTGAACCCCCAAACCTGACTGACTGGGCCAGTGTGCTGCAAATTGGCAGGAGACGTGAAG2040
GCACCTCCAAAGTCGGCCAAAATGAATGGGCAGTGAGCCGGGGTTGCCTGGAGCCGTTCC2100
TGCGTGGGTTCTCCCGTCTTCCGCTTTTTGTTGCCTTTTATGGTTGTATTACAACTTAGT2160
TCCTGCTCTGCAGATTTTGTTGAGGTTTTTGCTTCTCCCAAGGTAGATCTCGACCAGTCC2220
CTCAACGGGGTGTGGGGAGAACAGTCATTTTTTTTTGAGAGATCATTTAACATTTAATGA2280
ATATTTAATTAGAAGATCTAAATGAACATTGGAAATTGTGTTCCTTTAATGGTCATCGGT2340
TTATGCCAGAGGTTAGAAGTTTCTTTTTTGAAAAATTAGACCTTGGCGATGACCTTGAGC2400
AGTAGGATATAACCCCCACAAGCTT2425 (2) INFORMATION FOR SEQ ID NO:2: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 559 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: RNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:2:
GGGUUGCGGAGGGAGGGUGGGCCUGGGAGGGGUGGUGGCCAUUUUUUGUCUAACCCUAAC60
UGAGAAGGGCGUAGGCGCCGUGCUUUUGCUCCCCGCGCGCUGUUUUUCUCGCUGACUUUC120
AGCGGGCGGAAAAGCCUCGGCCUGCCGCCUUCCACCGUUCAUUCUAGAGCAAACAAAAAA180
UGUCAGCUGCUGGCCCGUUCGCCCCUCCCGGGACCUGCGGCGGGUCGCUGCCCAGCCCCC240
GAACCCCGCCUGGAGGCCGCGGUCGGCCGGGGCUUCUCCGGAGGCACCCACUGCCACCGC300
GAAGAGUUGGGCUCUGUCAGCCGCGGGUCUCUCGGGGGCGAGGGCGAGGUUCACCGUUUC360
AGGCCGCAGGAAGAGGAACGGAGCGAGUCCCGCGCGCGGCGCGAUUCCCUGAGCUAUGGG420
ACGUGCACCCAGGACUCGGCUCACACAUGCAGUUCGCUUUCCUGUUGGUGGGGGGAACGC480
CGAUCGUGCGCAUCCGUCACCCCUCGCCGGCAGUGGGGGCUUGUGAACCCCCAAACCUGA540
CUGACUGGGCCAGUGUGCU559 (2) INFORMATION FOR SEQ ID NO:3: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 1259 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:3:
TTTTTTTTTTCCTCGTAATCTTTTTTTTTGTTTTAAACACTGGAACTTGATGTCTGGAGG60
ACGGAGTCGGAGGATGTTCGACCCTAATATCCGAGCCCAGTCGATGGGAACTTTAAGAAA120
AAGAAAGACCTTGAGTCATGGACCAACCGGTACGTGAGTGTTCTCTAGGCGGACGGAAGA180
CAGTTTAAGACCTTAATTTCTAAACGCGGTGAAAAGGGGTGAAGGTGGGGGCCGACACCC240
TCACCTGACCCAACTTCCACCTTAAAAAAAAAAAAAAAAAAATCACTTTTTTCCCCCCTA300
ACCTTTATAGGGGATGAAATATCCTACTTTCAACTCTAGTATATTTCAGAAACCAAGCCT360
CAGAGATGTGCGTGCGTGCGTGTGTGTGTGTGTATGTGTGTGTGTCTCACAGCAAGAAAC420
AGATTTTATTATTTATTTTTTATTTATTTATTTTTTGCAAGTGACTGGCTAGGAAGAGTG480
GGGAAGCGGGAGGACAAATGGGGAAGAGGGAGCATTTCCGCAAGTGCTGGGCTCGACCAA540
TCAGCGCGAGCCATGGGGTATTTAAGGTCGAGGGCGGCTAGGCCTCGGCACCTAACCCTG600
ATTTTCATTAGCTGTGGGTTCTGGTCTTTTGTTCTCCGCCCGCTGTTTTTCTCGCTGACT660
TCCAGCGGGCCAGGAAAGTCCAGACCTGCAGCGGGCCACCCGGCGTTCCCGAGCCTCAAA720
AACAAACGTCAGCGCAGGAGCTCCAGGTTCGCCGGGAGCTCCGCGCGCCGGGCCGCCAGT780
CCCGTACCCGCCTACAGGCCGCGGCGCTGGGGTCTTAGGACTCCGCTGCCGCCGCGAAGA840
GCTGCGCTCTGTCAGCCGCGGGCGCGCGGGGCGTGGGGCAGGCGGGCGAGCGCGCGAGGA900
CAGGAATGGAACTGGTCCGTGTTCGGTGTCTTACTGAGCTGTGGGAAGTGCACCGGACTC960
GGTTCTCACACCCCATTCCCGCTGGGGAAATGCCCCGCTGCAGGGCGGGCCGCTAGAACC1020
TGCGACTCTGGGGAAAGGGGCTTCGGTGTGAGACGGTAGCCAGCCAAAGGGTATATATCG1080
CCCTCACGCCCCGTCCCCCTCCACTTTTGTCTAATACTCCTGTTTCTGTTGTGCAGATTT1140
TGCAGGCGTTTCGCTGGCTCTGCCTGAACGAGCTATCAGCCATGTGGTCCTTGGGGGTGG1200
GGGTGGGGATGGGTTGTGTAGTGCTGGGAATGAACCTAGTTTCTAAGTTCTCTATCAAC1259 (2)
INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 534 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:4:
CUCGACCAAUCAGCGCGAGCCAUGGGGUAUUUAAGGUCGAGGGCGGCUAGGCCUCGGCAC60
CUAACCCUGAUUUUCAUUAGCUGUGGGUUCUGGUCUUUUGUUCUCCGCCCGCUGUUUUUC120
UCGCUGACUUCCAGCGGGCCAGGAAAGUCCAGACCUGCAGCGGGCCACCCGGCGUUCCCG180
AGCCUCAAAAACAAACGUCAGCGCAGGAGCUCCAGGUUCGCCGGGAGCUCCGCGCGCCGG240
GCCGCCAGUCCCGUACCCGCCUACAGGCCGCGGCGCUGGGGUCUUAGGACUCCGCUGCCG300
CCGCGAAGAGCUGCGCUCUGUCAGCCGCGGGCGCGCGGGGCGUGGGGCAGGCGGGCGAGC360
GCGCGAGGACAGGAAUGGAACUGGUCCGUGUUCGGUGUCUUACUGAGCUGUGGGAAGUGC420
ACCGGACUCGGUUCUCACACCCCAUUCCCGCUGGGGAAAUGCCCCGCUGCAGGGCGGGCC480
GCUAGAACCUGCGACUCUGGGGAAAGGGGCUUCGGUGUGAGACGGUAGCCAGCC534 (2)
INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 569 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:5:
TGACTATTAGGGCTCAGCCAATCAGCGCGAGCTGTCGGGTATTTAGGGACGGGTATTTAG60
GGACAAGGGCCGCGCGACTTCTGCGTCTAACCCTATTGTTATAGCTGTGGGTTCTGTTCT120
TTTGTTCTCCGCCCGCTGTTTTTCTCGCTGACTTTCAGCGGGCCTGGAAAGTTCAGACCT180
GCAGCGGGTCACCGCGCATTCTGGACCTCAAAAAATGTCAGCGTAGGAAGCTCTGGTGCC240
AGAGCTCCGCGGCGCTGGGCCCGCCAGCCCGGTACCCGCCTGGAGGCCGCGGACGGCCTG300
GGGTCTTAGAACTCCGCTGCCGCCGTGAAGAGCTAGTCTCTGTTAGCTACGGGGCACCGG360
GCGCTGGGGTCAGGCCGGGAGAGCGCCGCAAGGACAGTAACGGAACTGGTCCCTGAGTTC420
GGTGGCTTTCCTGAGATGTGGGAAGTGCACCTGGAACTCAGTTCCTACAACCCCCACTTC480
CGCTGGGAAATGCCTTGCTACCTGGCGGGGCGCTAGAACTGCAACCGGGAGGAACGGGGC540
CAAGGTGTGTGCACGAGGCCACGGTGCTC569 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:6: CUCAGUUAGGGUUAGACAAA20 (2)
INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CGCCCUUCUCAGUUAGGGUUAG22 (2) INFORMATION FOR SEQ ID NO:8: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:8: GGCGCCUACGCCCUUCUCAGUU22 (2)
INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CAGGCCCACCCTCCGCAACC20 (2) INFORMATION FOR SEQ ID NO:10: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:10:
UAGGGUUACUGAUGAGUCCGUGAGGACGAAACAAAAAAU39 (2) INFORMATION FOR SEQ
ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
UUAGGGUCUGAUGAGUCCGUGAGGACGAAAGACAAAA37 (2) INFORMATION FOR SEQ ID
NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
UCUCAGUCUGAUGAGUCCGUGAGGACGAAAGGGUUA36 (2) INFORMATION FOR SEQ ID
NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
CCCGAGACUGAUGAGUCCGUGAGGACGAAACCCGCG36 (2) INFORMATION FOR SEQ ID
NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ix) FEATURE: (A) NAME/KEY: misc.sub.-- feature (B) LOCATION: 20
(D) OTHER INFORMATION: /note= "Amino blocking group linked at this
position." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
ATAGCGGCCGCAAGAATTCA20 (2) INFORMATION FOR SEQ ID NO:15: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:15: TGAATTCTTGCGGCCGCTAT20 (2)
INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:16: GGAAGGTCTGAGACTAG17 (2) INFORMATION FOR SEQ ID NO:17: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:17: ATCTCCTGCCCAGTCTG17 (2)
INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:18: GAGAAAAACAGCGCGCGGGGAGCAAAAGCA30 (2) INFORMATION FOR SEQ ID
NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GTTTGCTCTAGAATGAACGGTGGAAG26 (2) INFORMATION FOR SEQ ID NO:20: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:20: GCCTGGAGGGGTGGTGGCCATTTTTTG27
(2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:21: GATCGGCGTTCCCCCCACCAAC22 (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:22:
CGTCGACTAGGGTCGAGGGCGGCTAGGCCT30 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:23:
GGAGGCGGCCGCAGACGTTTGATTTTTTGAGGC33 (2) INFORMATION FOR SEQ ID
NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GGAGGCGGCCGCAGACGTTTGTTTTTTGAGGC32 (2) INFORMATION FOR SEQ ID
NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: ATGAAAATCAGGGTTAGG18 (2)
INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:26: CCACAGCTAATGAAAATC18 (2) INFORMATION FOR SEQ ID NO:27: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:27: CCACAGCTAATGAAAATCAGGGTTAGG27
(2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:28: TCACGTTCAAGGGTTAGG18 (2) INFORMATION FOR SEQ ID NO:29: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:29: ATGAAAATCGCTACCTAA18 (2)
INFORMATION FOR SEQ ID NO:30: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:30: CCCACAGCTAATGAAAAT18 (2) INFORMATION FOR SEQ ID NO:31: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:31: CCCCACAGCTAATGAAAA18 (2)
INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:32: CACCGGGTTGCGGAGGGAGG20 (2) INFORMATION FOR SEQ ID NO:33: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:33: GGAGGGGCGAACGGGCCAGCA21 (2)
INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:34: CGTCGACTAGCGCTGTTTTTCTCGCTGACT30 (2) INFORMATION FOR SEQ ID
NO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
GGAGGCGGCCGCAGGTGCACTTCCCACAGCTCAG34 (2) INFORMATION FOR SEQ ID
NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: CUAACCCUAAC11 (2)
INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ix) FEATURE: (A) NAME/KEY:
modified.sub.-- base (B) LOCATION: 11 (D) OTHER INFORMATION:
/mod.sub.-- base=i (ix) FEATURE: (A) NAME/KEY: modified.sub.-- base
(B) LOCATION: 12 (D) OTHER INFORMATION: /mod.sub.-- base=i /note=
"Inosine is linked with biotin" (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:37: UUAGGGUUAGNN12 (2) INFORMATION FOR SEQ ID NO:38: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ix)
FEATURE: (A) NAME/KEY: modified.sub.-- base (B) LOCATION: 11 (D)
OTHER INFORMATION: /mod.sub.-- base=i (ix) FEATURE: (A) NAME/KEY:
modified.sub.-- base (B) LOCATION: 12 (D) OTHER INFORMATION:
/mod.sub.-- base=i /note= "Inosine is linked with biotin" (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:38: AUUGGGUUAUNN12 (2) INFORMATION
FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 base
pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: CTAACCCTA9 (2)
INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 9 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:40: CAAACCCAA9 (2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 10 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:41: CCAACCCCAA10 (2) INFORMATION FOR SEQ ID
NO:42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: CTCACCCTCA10 (2)
INFORMATION FOR SEQ ID NO:43: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 552 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:43:
GCGAGAGCCGGCGCCGGCCAATCAGCGCGCGCCACCCCGGGTACTTAAGGGCGACCTGGC60
GGGCGGCTGCCAGTCTAACCCTGAATTCTGAGAGCTGTGGGTACTGTGCTTTCGTCTCCG120
CCCGCTGTTTTTCTCGCTGACTTCCAGCGGGCGGGAAAGTCCAGACCTGCAGCGGGCCAT180
CGCGCGTTTTCCACCACAAAAAAATGTCAGCGCTGGCGTCATGTGCCTGGAGCCTTGCGC240
CGGCCCGCCAGCCCCGCACCCGCCTGAGGCCGCGGTCGGCTGGAGTCCTCGGGCTCCGCT300
GCCGCCGCGAAGAGCTAGACTCTGTCAGCCGCGGGGCGTCAGGGGCTGGGGCGAGCCGGC360
AGCGCCGCAAGCAGAGAAACGGAGCTGGTCCCGTGAACGGTGACTTCCCTGAGTTGTGGG420
AAATGCACCAGGAACTCGGTTCCCACAACCCCCAACCCCGCTGGGAAATAACCTGCTGCA480
AAGCGGGCCCCTAGGACCTGGCAGCCCGAGGAATGGTGCCAACGTGTGTGCACATGGCCA540
GAGTGGGCGATG552 (2) INFORMATION FOR SEQ ID NO:44: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 590 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
CAGCCTTCAAAAATGAGGAGATCCGGGTTGCGGAGGGTGGGCCCCGGGTTGGGTGGGCCC60
CGGGTTGGTGGCAGCCATTTCTCATCTAACCCTAATTGAGACAGGCGTAGGCGCTGTGCT120
TTTGGTTACCGCGCGCTGTTTTTCTCGCTGACTTTCAGCGGGCGGAAAAGCCTCGGCCTA180
CCGCCATCCACCATCCAGTCTGCAACAAACAAAAAATGTCAGCCGCTGGCTCGCTCACCT240
CTCCCGGGAACCTGCGGTGGTCCGCCCGCCCAGCCCCAGTGCCCCGCCTGAGGCCGCGGT300
CGGCCGGGGCTTCTCCGGAGGCACCCATTGCCGCCGTGAAGAGTTGGGCTCTGTCAGCCG360
CGGGTCGCTCGGTGGGCCGAGGCATGGCTGTAACCGCAGGGAAAGGAACGGAGTGGGGTC420
CCCGCGCGCGTGCGTTCCCTGAGCTGTGGGACTTGCACCCGGGACTCGGCTCAGACATCT480
GAAAAAAAAAAAAATGAGGAGATCCTACCATATGAAACAATATGAACAAAACTTGAGGTT540
GTGCTAAGTGAAGTAAGTCAGCCATAGAAGGACAAATACTGTTACAATTC590 (2)
INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 580 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: RNA (genomic) (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:45:
CUCGACCAAUCAGCGCGCGCCAUGGGGUAUUUAAGGUCGAGGGCGGCUAGGCCUCGGCAC60
CUAACCCUGAUUUUCAUUAGCUGUGGGUUCUGGUCUUUUGUUCUCCGCCCGCUGUUUUUC120
UCGCUGACUUCCAGCGGGCCAGGAAAGUCCAGACCUGCAGCGGGCCACCCGGCGUUCCCG180
AGCCUCAAAAACAAACGUCAGCGCAGGAGCUCCAGGUUCGCCGGGAGCUCCGCGGCGCCG240
GGCCGCCCAGUCCCGUACCCGCCUACAGGCCGCGGCCGGCCUGGGGUCUUAGGACUCCGC300
UGCCGCCGCGAAGAGCUCCGCCUCUGUCAGCCGCGGGCGCGCGGGGGCUGGGGCCAGGCC360
GGGCGAGCGCCGCGAGGACAGGAAUGGAACUGGUCCCCGUGUUCGGUGUCUUACCUGAGC420
UGUGGGAAGUGCACCCGGAACUCGGUUCUCACAACCCCCAUUCCCGCUGGGGAAAUGCCC480
CGCUGCAGGGCGGGCCGCUAGAACCUGCGACUCUGGGGAAAGGGGCUUCGGUGUGAGACG540
GUAGCCAGCCAAAGGGUAUAUAUCGCCCUCACGCCCCGUC580
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